WO2018229622A2 - System for simultaneous electrophoresis in multiple gels and interpretation of results with permanent standard ladders - Google Patents

Abstract

The present inventions relates to a system for compartmentalizing of the cavity of electrophoresis apparatus for simultaneous electrophoresis of samples in gels of different concentrations to find out which concentration gives better separation and resolution. This system is comprised of a comb with detachable teeth and separators. The separators used in place of detachable teeth in appropriate slots on the comb gives compartmentalization. Gels of different concentrations can be simultaneously set, one in each compartment, followed by loading of samples in the wells and electrophoresis run. One more aspect of the invention is a permanent standard ladder or 'gelcard' specific for a gel type and concentration used for estimation of the base pair length of nucleic acid samples or molecular weight of protein sample. 'Gelcard' can be usedonly if the electrophoresis conditions of sample run are same to those during preparation of the 'gelcard'.

Description

TITLE - SYSTEM FOR SIMULTANEOUS ELECTROPHORESIS IN MULTIPLE GELS AND INTERPRETATION OF RESULTS WITH PERMANENT STANDARD LADDERS

FIELD OF INVENTION- Present invention belongs to the field of biotechnology, molecular biology, instrumentation and techniques which particularly relates to improvement in electrophoresis apparatus to allow simultaneous runs of gels of multiple concentration at one go and estimation of nucleic acid or protein bands resulting from electrophoresis using a permanent and reusable detection aid. BACKGROUND: -Gel electrophoresis is the method of separating charged bio- molecules through a gel matrix under the influence of electric current. Among the various parameters, molecular weight of the biomolecule and gel pore size are the main decisive factors that help in differentiating among nucleic acids and proteins in a given mixture during gel electrophoresis. Majorly there are two types of gel electrophoresis, Agarose gel electrophoresis (AGE) and Polyacrylamide Gel Electrophoresis (PAGE); the former used for differentiating nucleic acids and latter for both nucleic acids and proteins. Apart from differing in the linkage units that make up the gel (agarose units for AGE; polyacrylamide for PAGE) they differ in many aspects. PAGE is tedious with more expertise required in its preparation than AGE owing to two reasons; one being that the polymerisation is rapid in PAGE so the gel has to be casted immediately, secondly the gel size is equivalent to the space between two glass slides created by the spacer is minimal in comparison to AGE, thus pouring and casting a smooth gel of minimum thickness without any bubbles or irregularity requires precision and steady hand. With respect to the above point, the volume of gel in PAGE is comparatively less with maximum being about 15ml, whereas barring smaller gel trays, the minimum volume for AGE would be about 25ml.

During gel electrophoresis, the DNA and proteins are separated out as bands with higher molecular weight/base pair length bands found nearer to the well and the lower molecular weight ones towards the end of the 'run' [Fig. l].The confirmation of the identity in the capacity of a band is verified using a ladder, for nucleic acid samples DNA/RNA ladders are used and for protein samples protein ladders are used. A ladder is defined as a double-stranded or single- strandedDNA/RNA or protein fragments whose molecule weights (corresponding to their amino acid content) or fragment size (corresponding to their base pair content) are known and standardized. A particular ladder comprises of mixture ofDNA or RNA of known base pair (bp) or proteins of known molecular weight (M.wt) depending on the ladder. As a routine practice, a ladder is 'run' along with the sample in separate well, the ladderaids in defining the M.wt/bpof the band thus allowing the identification of the DNA/RNA/Protein fragment of interest.

These standard ladders are quiet costly. A researcher selects the ladder based on the fact the ladder contains the Mwt/bp band which exactly defines the band of his/her interest. For example, if one has to verify that a PCR amplicon as 5000bp, he/she prefers a lkb ladder (Thermoscientific, Gene Ruler 1 kb DNA Ladder, 0.5 μg/μL) which has 14 bands corresponding to 250bp, 500bp, 750bp, lOOObp, 1500bp, 2000bp, 2500bp, 3000bp, 3500bp, 4000bp, 5000bp, 6000bp, 8000bp and 10,000bp based on the order of their positioning from bottom of the gel towards the well. When the sample is run alongside the 1Kb ladder, the sample band is found to align aligns exactly at the same position that of the 5000bp within the ladder [Fig 2]. For every possible bp/M.wt there are no unique or customised ladder available, in such instances the molecular weight or base pair ladder gives guesstimate of the molecular weight (M.wt) or base pair (bp) of the 'band' under study rather than the exact M.wt/bp. To figure out a nearly appropriate M.wt, comparison of the candidate band against two ladders is another option. For example, if the researcher is to confirm a DNA fragment of about 475bp, he /she havetwo options. In one, run with just running a lOObp ladder and figure out that the band will be nearer towards500bp or with 25bp ladder such that the band falls between 450 bp and 500bp. In the second option, run two ladders [Fig 3] of lOObp and 25bp ladder each, such that with both one can estimate a better positioning and defining of the 475bp. Citing either first or second scenario, one or two wells in the AGE is slotted for the ladder alone, so if one has 10 samples to compare and have only a 10 well AGE gel, the researcher has to run it in two gels and both times ladder has to be run resulting in wastage of resources and excess time for pre and post preparation of AGE. The same scenario applies for PAGE as well.

For prepration of polyacrylamide gel, a front glass slab which is rectangular in shape [Fig. 4(a)] and a back glass slab which is rectangular in shape with a notch on the upper side to hold the comb [Fig. 4(b)] along with two spacers [Fig 4(c)], one at left hand side and one at right hand side. After placing the spacer on the left and right side within the two glass slabs, this assembly is fixed together with an adhesive tape or by clamps depending on the electrophoresis unit. The thickness of the gel is guaranteed by the spacer that are placed on left and right side of the slabs. The bottom is sealed with agrose solution. After gelation of agarose, resolving gel is prepared and carefully introduced between glass slabs upto 70% of the available length and allowed to polymerise. This followed by introducing the stacking gel ofsame polyacrylamide concentration but with different pHon the top of resolving gel and placing the comb [Fig 4(d)] to create wells[Fig 4(e)]. The resolving gel topped with stacking gel is used for electrophoresis of proteins only. In case of nucleic acid one single gel or resolving gel is used. Polymerisation around the comb results in creation of wells. The samples mixed with loading dye are loaded in different wells along with a standard ladder solution as a reference in a seperatewell. Both the standard and samples are allowed to run under electric current. After the electrophoretic run when the dye front have covered three fouth the distance, the supply of electric current is stopped. The slabs are removed and polyacrylamide gels are stained in coomasssie blue for 2 hours followed by overnight destaining in destaining solution in case of protein samples. The light blue bands are then visualized. In case of nucleic acid samples, the gel is first washed with TAE buffer and then stained for 30 minutes with ethidium bromide. Instead of ethidum bromide, sometimes other stains are also used. The gel slab is once again rinsed with TAE buffer and then the bands are visualized under UV light.

The limitation of this technique is that, at one go, a user has to carry out an experiment with only one specific concentration of polyacrylamide. Secondly, if there are only one or two samples to 'run', rest of the well spaces are usually wasted. Thirdly, for every ladder run the well is occupied, so in cases where the sample number is exact to that of the teeths in the comb, a separate gel has to be made to run the extra sample along with a ladder which leads to extra labour and use of resources. For preparation of Agarose Gel for electrohoresis, gel casting tray is used[Fig 5(a)]. As the preliminary step, the open ends of the casting tray are sealed with cellotape and it is checked for leakage by pouring water into it. Required amount of Agarose is added to electrophoretic buffer and heated till the agarose is competely dissovled to achieve a specific concentration of agarose. Then required amount of ethidium bromide is added to it and mixed well. Ethidium bromide is an intercalating agents thatintercalates between the basepairs and fluoresce under the UV light. The comb is placed on the casting tray and the warm agarose is then poured in the casting tray. The gel is allowed to settle with the comb in place for creation of wells into the gel[Fig 5(b)]. The wells are used for loading nucleic acid samples that are 'run' through the gel matrix under electric current. Nucleic acids being negatively charged, migrate to the anode from cathode. For every run, it is a standard practice to reserve one well for standard or molecular ladder. The ladder comprise of DNA/RNA molecules of known base pair lengths are loaded which could help in guestimating the DNA or RNA of the samples and help in defining the identity of the sample. During electrophoresis, the nucleic acids are seperated based on their molecular weight. The number of basepairs decide the molecular weight of the samples, lesser the basepairs, lesser the length so lesser the molecular weight. If a sample comprising mixed amount of small, medium and larger molecular weight nucleic acid samples, during electrophoresis, the run of smaller fragments is more than the medium followed by larger. The nucleic acids with similar molecular weight move together through the gel matrix pores such thatthey appear as bands during visualisation under UV ight. Another practice during every gel run is mixing loading dye with each samples. The dye runs along with the sample towards the cathode which helps in ease of visualising and keeping track of the electrophoretic run, commerically avaialble molecular ladder come mixed with loading dye. Usually theelectrophoretic run is stopped when the loading dye front covers almost three fourth of the gel tray distance. The ethidium bromide stained gel is taken out and exposed to UV light usingUV transilluminator. This exposure causes nucleic acid bound ethidium bromide to fluoresce and nucleic acids to appear as bands [Fig 5(c)].

Specific concentration of polyacrylamide/agarose gives rise to a specific pore size of the resultant gel, which is the decisive factor in separation and resolution of peptide bands/DNA(or RNA) bands. The usually used concentrations of polyacrylamide are 8%, 10%, 12% and 15% , whereas in case of agarose, concentrations are 0.8%, 1%, 1.5% and 2%. To guarantee proper resolution, samples are run at different gel concentrations of different pour sizes. For every run, the ladder is also used to track the base pair or molecular size of the sample of interest. In such scenario for every ladder, a well for sample is used up. Thus for multiple samples, more gels are cast to faciliate both sample and ladder run resulting in more usage of gel preparing chemicals, labour and time. In case the comparison is for single or few nucleic acid or protein samples, multiple gels which differing concentration have to be prepared as there is no provision to run multiple concentration of gels in the sample casting tray. This practice result in increased usage of chemicals, labour, time as well as sample and ladder resources apart from non-optimal usage of the entire wells in the casted gel.

Another unforeseen difficulty in gel elctrophoresis is the quality of ladder. Continous freezing and thawing of the ladder causes distortion and denaturation of the bands thatinfluence the electrophoretic migration. In such scenario the gel has to be casted again and steps repeated with a new ladder. In other cases, manual pipetting error results in limited amount of ladder in the gel such that visualisation and intepretation of the bands with respect to ladder is impossible resulting in yet another fresh run. These events increases the time and cost factor involved in the experimentation, many folds. When samples are run in multiple gel trays, comparitive analysis of the bands without ladder can be brought about if all the gel are uniformly run. This is guaranteed by allowing the same run distance of the samples based on the elctrophoretic run of the loading dye. A perfect comparison of bands is posssible when one gel picture is aligned over multiple gel runs containing different samples.

A solution to guarantee uniform gel run with limited resources and optimal use of gel and the wells is required.

DEFINITIONS

1. PAGE - PolyAcrylamideGel Electrophoresis is an electrophoretic method of separating nucleic acids and or proteins based on their charge and molecular weight (Native PAGE) or solely on their molecular weight (SDS-PAGE). The copolymerisation or crosslinks between poly-acrylamide and bis-acrylamideis made possible by free radicles generated from Ammonium per sulphate (APS) which is triggered by Tetramethylethylenediamine (TEMED) that act as a catalyst. The Polyacrylamide gel mesh thus formed comprises of pores due to the cross-linkages, these pores allow the molecules to pass through under the influence of electric current and thus biomolecules are separated.

2. AGE - Agarose Gel Electrophoresis is an electrophoretic method of separating biomolecules based on their molecular weight and conformation. Even though both protein and nucleic acids could be separated, AGE is preferably used for nucleic acids (DNA and RNA) separation. The agarose gel is formed by gelation through hydrogen bonding and electrostatic interactions of agarose. The biomolecules are separated under the influence of electric current through the pores within the agarose matrix formed during gelation. 3. Standard Ladder - is a marking or approximating entity comprising of protein or nucleic acid molecules of different sizes that help in guesstimating molecular weight or base pair size of protein or nucleic acids. These are also termed as molecular markers. Standard markers come in different combinations with protein and nucleic acids of varying sizes which act as a scale to define the molecular size of the sample protein and or DNA. The user can choose from a catalogue of protein ladders and DNA ladders the optimal one depending on the fragment size they need to identify.

OBJECTS One objective of current invention is to provide a means for separating the cavity between glass slabs of PAGE assembly as well as cavity of gel casting tray of AGE into multiple compartments where the means is a separator which can be attached to the comb used for creating wells.

The other objective of the current invention is to provide a modified comb which contains multiple tooth holders where each tooth holder is capable of holding a separator as well as a tooth, but only one at a time.

One more objective of the current invention is to provide a means which replaces the need of running 'standard peptide ladder' and standard DNA/RNA ladder during each run of electrophoresis in the form of a collection of strips where each strip i.e. a 'gelcard' or 'laddercard', is inscribed with standard peptide bands or DNA/RNA fragment size marking sunder a specific concentration of polyacrylamide/agarose components. One band on a strip corresponds to a band of a peptides/nucleic acids of specific molecular weight/base pair size

Yet one more objective of the invention is to provide a method which uses the separators along with comb to create multiple gels, each of a specific concentration within one assembly of AGE/ PAGE and use them simultaneously at one go to run a sample on AGE/ PAGE of different concentration with circumvention of the need of running 'standard peptide / DNA (or RNA) ladder for gel of each concentration and interpretation of the molecular weight/ base pair length of the bands obtained in samples by comparing them with 'gelcards' of the current invention.

SUMMARY

The disclosed invention a gel electrophoresis apparatus having modified comb comprising a buffer tray to hold the electrophoretic buffer in said apparatus; either glass slabs with side spacers making an inner cavity or a gel casting tray having a space for setting and holding of the electrophoretic gel in said apparatus; an anode and a cathode for electric supply; a modified comb to accommodate detachable teeth and separators within it. One aspect of the invention is the said modified comb essentially consisting of, a rectangular planar body(600)having alternating portions one being a basal portion tooth-spacer (603 B) having means of attachment for attaching detachable teeth (604)and another being an elevated portion tooth-holder (603 A), two side edges for resting said modified comb on said side spacers or said gel casting tray, detachable teeth (604) having means of attachment for getting attached with said tooth-spacer portions, an attachment tool (607) for attaching said detachable teeth (604) with said tooth-spacer (603 B),and one or more separators (606) for compartmentalizing said inner cavity of the glass slabs or said space of gel casting tray for setting and holding multiple gels within the said apparatus, wherein the tooth-spacer is the basal portion of thickness and width appropriate to accommodate a tooth or a separator and the tooth-holder is the elevated portion of thickness and width appropriate for supporting and placement of tooth or separator in adjacent tooth-spacer.

Another aspect of the invention is an artificial permanent ladder for identification and analysis of nucleic acids using gel electrophoresis comprising a rectangular shaped travel -distance scale having a top portion having marking indicating the start of gel run; a bottom portion having marking indicating the stop of gel run; and a middle portion marked with indications at various distances which corresponds to base pair lengths of specific nucleic acids, wherein said correspondence relation between the indications and the base pair lengths are derived after running more than one gel electrophoresis experiments for nucleic acids of known base pair lengths under controlled conditions, measuring the distances travelled through gel by said nucleic acids, and marking permanent indications at same distances on said artificial rectangular shaped travel-distance scale of length equivalent to said gel to denote the start of gel run, the stop of gel run, and the specific base pair lengths.

One more aspect of the invention is An artificial permanent ladder for identification and analysis of proteins using gel electrophoresis comprising a rectangular shaped travel -distance scale having a top portion having marking indicating the start of gel run; a bottom portion having marking indicating the stop of gel run; and a middle portion marked with indications at various distances which corresponds to molecular weights of specific proteins, wherein said correspondence relation between the indications and the molecular weights are derived after running more than one gel electrophoresis experiments for proteins of known molecular weights under controlled conditions, measuring the distances travelled through gel by said proteins, and marking permanent indications at same distances on said artificial rectangular shaped travel -distance scale of length equivalent to said gel to denote the start of gel run, the stop of gel run, and the specific molecular weights.

Yet one more aspect of the invention is a method of running simultaneous electrophoresis for nucleic acids or proteins in a single electrophoresis apparatus in gels of different concentrations comprising a) arranging an electrophoresis apparatus having following components - buffer tray to hold the electrophoretic buffer in said apparatus; either glass slabs with side spacers making an inner cavity or a gel casting tray having a space for setting and holding of the electrophoretic gel in said apparatus; an anode and a cathode for electric supply; a rectangular planar body (600) having alternating portions one being a basal portion tooth-spacer (603 B) with thickness and width appropriate to accommodate a detachable tooth (604) or a separator (606) and having means of attachment for attaching detachable teeth (604), and another being an elevated portion tooth-holder (603 A), and

- two side edges for resting said rectangular planar body on said side spacers or said gel casting tray; - one or more separators (606) for compartmentalizing said inner cavity of the glass slabs or said space of gel casting tray for setting and holding multiple gels within the said apparatus; and b) attaching detachable teeth (604) to said rectangular planar body (600) and attaching one or more said separators(606) to said rectangular planar body(606), resting said rectangular planar body on said side spacers and placing the said separators at required positions within inner cavity bound by said glass slabs and removing the rectangular planar body with attached said detachable teeth or attaching detachable teeth (604) to said rectangular planar body (600) placed within said gel casting tray and aligning the said separators placed within the cavity of gel casting tray with said empty tooth- spacers on the said rectangular planar body and removing the rectangular planar body with attached said detachable teeth; c) adding gels of varying concentrations in different compartments created by compartmentalizing said inner cavity of the glass slabs or said space of gel casting tray; d) resting the said rectangular planar body (600) with attached said detachable teeth said side spacers or said gel casting tray within dipping of attached teeth in the gels multiple compartments in said inner cavity of the glass slabs or said space of gel casting tray; e) allowing said gels of varying concentrations to set in said compartments with creation of wells around detachable teeth and thereafter removing said rectangular planar body (600) along with the detachable teeth attached to it from the electrophoretic apparatus; f) introducing the nucleic acid or protein in the wells created in gels around detachable teeth; g) starting the electric supply for running simultaneous electrophoresis of nucleic acid or protein in gels of different concentrations in said single electrophoresis apparatus; and h) analysing the obtained bands in various gels by using suitable detection techniques.

BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWINGS Fig 1: Illustrates a gel slab(100) with wells in it(101), with a band of high molecular weight peptide/Nucleic acid (102) nearer to the loading well and a band of low molecular weight peptide/Nucleic acid (103) farther from the loading well

Fig 2:- Illustrates a gel slab(200) with four wells in it (201 to 204), with solution of known fragments, standard (ladder) loaded in well 201 and three samples loaded in wells 202 to 204. The separation of standards in the form of ladder of bands (205) along with matching of one sample band with one within the standard bands in ladder. Band shown in 202 matches with 4^th band from bottom of ladder (205); band from the well 203 lies between 3^rd and 4^th band of ladder (205) from the top; band of well 204 matches with 6^th band of ladder (205) from the bottom.

Fig 3:- Illustrates a gel slab(300) with four wells in it(301 to 304), with solutions of two standards loaded in wells 301 and 304 respectively and two samples loaded in wells 302 and 303 and separation of standards in the form of ladders(305 and 306), where bands of ladder 305 are of broad range and matches approximately with sample bands and bands of ladder 306 are of narrow range matches exactly with sample bands. Fig 4(a) :- Illustrates BackGlass Slab used for construction of cavity for preparation of Polyacrylamide Gel

Fig 4(b) :- Illustrates Front Glass Slab used for construction of cavity for preparation of Polyacrylamide Gel with a notch 401 used for placing comb for creating of wells in gel Fig 4(c) :- Illustrates spacers which are placed between front glass slab and back glass slab on extreme left and right side to construct a cavity of the depth equal to thickness of separator in which solution of polyacrylamide composition is introduced which polymerises into gel.

Fig 4(d) :-Illustrates a comb which is used to create wells for loading samples in gel

Fig 4(e) : -Illustrates final assembly of all componets used to create polyacrylaminde gel where 402 is back glass slab, 403 is front glass slab, 405 are spacers placed between front and back glass slab on right hand edges and lenft hand edges, 404 is the comb placed in notch of front glass slab for creation of well and 406 is the boundry of resolving gel and stacking gel

Fig 5(a) - Illustrates casting tray of Agarose Gel Electrophoresis.

Fig 5(b) - Illustrates casting tray of Agarose Gel Electrophoresis (503) sealed with cellotape (501) and containing agarose gel(504) with comb(502) in place for creation of wells Fig 5(c) - Illustrates nulceic acid bands of standard ladder and sample embeded in agarose which separated due to electrophoresis and showing fluorescence under UV light due to bound ethidium bromide. Fig 6(a) - Illustrates Comb body (600) with a backbone (601) with alternate tooth-holders(603A) and tooth-spacers(603B) with a set of three means of attachment on tooth-spacers(602) and a C shaped rester(603C).

Fig 6(b) - Illustrates a detachable tooth(604) with two sets of three means of attachment (605)

Fig 6(c) - Illustrates separator (605) for compartmentalization of AGE casting tray cavity

Fig 6(d) - Illustrates separator (606) for compartmentalization of cavity of PAGE assembly Fig 6(e) - Illustrates attachment tool (607) for fixing detachable teeth on teeth- spacers of the comb body

Fig 6(f) - Illustrates comb (600) with some teeth (604A) aligned to mean position with all three means of attachment of the lower set on a tooth are aligned with all the three means of attachment on the corresponding tooth spacer on the comb between two adjoing teethholders and attached with attachment tool (607) ; two teeth (604B, 604C) aligned in a similar way to a position upper to mean position (604B, 604C) , two teeth aligned in again a similar way to a position lower to mean position (604D, 604E).

Fig 7(a) - Illustrates a PAGE assembly within electrophoretic tray (707) with front glass slab(701), back glass slab(702) along with spacers (703) in between and a comb body(600) resting on spacers with detachable teeth attached using attachment tools (607)to fix the aligned means of attachment on teethspacer on comb body with means of attachment on detachable teethalong with a separator(606) placed on a toothspacer on comb body producing two compartments (704 & 705) containing two polyacrylamide gels of two different concentrations in the cavity bound by front and back glass slabs Fig7(b) - Illustrates a PAGE assembly of figure 7(a) with samples loaded in wells (708), and the whole assembly placed within electrophoreitc tray (707) filled with buffer (709) and supplied with electric current between cathode(705) & (706)

Fig 8- Illustrates an agarose gel assembly in the gel casting tray (901) divided into two compartments by the separator (907) containing gel slabs in each compartment of different agarose concentration (903 & 904) , with a comb (905) placed on the tray with detachable teeth (906) attached to it, with ends inserted inside gels in both compartments for well creation.

Fig 9 - Illustrates an agarose gel assembly undergoing electrophoresis comprised of a electrophoresis chamber (1001) in which gel casting tray (1002) containing two gels of different concentrations (1005 & 1006) separated by a separator (1004) is placed. Each gel contains wells which are loaded with samples (1007). Entire assembly is filled with electrophoresis buffer and electric current flows between cathode (1008) and anode (1009). Fig 10 - Shows a PAGE assembly(l 101) with a comb with detachable teeth(1103) and a separator (1104) resting upon side spacers(1102), with two compartments, one containing a polyacrylamide gel of concentration 8%(1105) and second containing a polyacrylamide gel of concetration 12%(1106), with ends of detachable teeth dipped within gels Fig 11- Showing three repeats of electrophoretic run of three samples and a 100 bp ladder in each repeat in 8% polyacrylamide gel.

Fig 12 - Showing three repeats of electrophoretic run of three samples and a 100 bp ladder in each repeat in 12% polyacrylamide gel.

Fig 13 - Showing an AGE casting tray(1201) with a comb with detachable teeth(1203) and a separator (1202) fixed within the cavity of casting tray at a position corresponding to fifth tooth-spacer of comb, agarose gel of 0.8% concentration in one compartment(1204) and agorse gel of 1.7 concentration in other compartment(1205), with the detachable teeth of comb dipped within gels. Fig 14(a) & Fig 14(c) - Illustrtes Front and Back of a 'gelcard' of 100 bp ladder in 1.7% Agarose

Fig 14(b) & Fig 14(d) - Front and Back of a 'gelcard' of 100 bp ladder in 1.0% Agarose Fig 15(a) - Shows Sample base pair comparison with front side of gelcard marked with indications for different base pair lengths, perecentage of polyacrylamide gel

Fig 15(b) - Shows Sample base pair comparison with back side of gelcard marked with different base pair lengths in number, perecentage of polyacrylamide gel

DESCRIPTION OF THE INVENTION : -

A molecular biology researcher has to run a sample for optimal separation and resolution in PAGE/ AGE. For the same the researcher may prefer to run it in different concentrations of Polyacylamide/ Agarose for accurate and reproducible results. During each cycle it is necessary that standards should also be run. This leads to usage of large quantities of costly chemicals and needs more time. So there is a need of mechanism which will allow to run the samples with different concentrations of Polyacrylamide/ Agarose and also cirucmvents repeated use of standards. The present disclosure relates to a gel electrophoresis apparatus having modified comb. The said electrophoresis apparatus comprises a buffer tray to hold the electrophoretic buffer in said apparatus; either glass slabs with side spacers making an inner cavity or a gel casting tray having a space for setting and holding of the electrophoretic gel in said apparatus; an anode and a cathode for electric supply; a modified comb to accommodate detachable teeth and separators within it. The said apparatus is used for gel electrophoresis of nucleic acid sample using a polyacrylamide gel electrophoresis or an agarose gel or gel electrophoresis of proteins using SDS polyacrylamide gel. One aspect of the disclosure is a modified comb which is having detachable teeth[Fig 6(a) & (b)]. One embodiment of the comb is its basic body [Fig 6(a)] which is roughly rectangular in shape and is comprised of continuous backbone - (601) [Fig 6(a)]on which elevated tooth-holders - (603A) [Fig 6(a)]and base level tooth-spacers -(603B) [Fig 6(a)] are present in alternate sequence. On the tooth- spacers a set of three means of attachment -(602) [ Fig 6(a)]is present to facilitate fixation of detachable teeth . A 'C shaped rester - (603C) [Fig 6(a)] is attached to the comb backbone just below the means of attachment and is perpendicular to the backbone of the comb. One end of this rester is attached on the left hand side just before start of first tooth-spacer and second end is attached on the right hand side just after the last tooth-spacer. This rester allows firm resting of the comb on the electrophoretic assembly.

The other embodiment of the modified comb are detachable teeth - (604) [Fig 6(b)], which are also rectangular in shape and each detachable tooth having two sets means of attachments -(605) [Fig 6(b)]where each set is comprised of three means of attachment. The detachable teeth (604) are of thickness appropriate to match the level of elevation of the tooth-holder, of width appropriate to fit into the tooth-spacer firmly and of length enough to dip in the gel set in the inner cavity of the glass slabs or in space of gel casting tray. The means of attachment of detachable teeth (604) and means of attachment of a tooth-spacer are (603 B) which are holes having equal diameters, and the detachable teeth (604) are attached to tooth-spacers (603 B) using the attachment tool (607) that percolate through said holes.

One detachable tooth can be attached to comb on one tooth-spacer by aligning means of attachment of a detachable tooth (604) and means of attachment of a tooth-spacer (603 B) which are holes having equal diameters, and attaching the detachable teeth (604) to tooth-spacers (603 B) using the attachment tool - (607)] [Fig 6(e)] which is a pin that percolate through said holes . Alternatively the means of attachment of detachable teeth (604) and means of attachment of a tooth-spacer (603 B) are glueable portions, and the detachable teeth (604) are attached to tooth-spacers (603 B) using an adhesive. Multiple teeth can be attached to the comb body in this manner based upon the requirements of number of compartments within the gel cavity and number of wells required in each compartment. Different lengths of teeth below the body of the comb can be achieved by choosing appropriate alignments of the means of attachments[Fig 6(f)].

The another aspect of the disclosure is a separator - (606) [Fig. 6(c) & Fig6(d)] of thickness, width and height appropriate to compartmentalize the inner cavity of the glass slabs or the space of gel casting tray. One or more separators can be used to create two or more compartments.

In case of PAGE, one or more seprarators (606) are placed on required toothspacers -(603 B) and detachable teeth(604) are fixed on the other toothspacers -(603 B) of the modified comb and placed within inner cavity of the glass slabs for compartmentalization [Fig 7(a)]. In case of AGE one or more seprarators (606) are placed within cavity of gel casting tray and are aligned with the empty toothspacers -(603 B) of the modified comb with detachable teeth(604) attached on the other toothspacers -(603 B) and fixed within the cavity of gel at aligned positions [Fig 8].

The modified comb including rectangular planar body, side edges, detachable teeth, and as well as the separators and attachment tool are made of a material inert to all reagents of polyacrylamide gel electrophoresis and agarose gel electrophoresis.

One more apsect of the disclosure is an artificial permanent ladder, 'gelcard', for identification and analysis of nucleic acids using gel electrophoresis comprising a rectangular shaped travel -distance scale having a top portion having marking indicating the start of gel run, a bottom portion having marking indicating the stop of gel run and a middle portion marked with indications at various distances which corresponds to base pair lengths of specific nucleic acids. The relation between the indications and the base pair lengths are derived after running more than one gel electrophoresis experiments for nucleic acids of known base pair lengths under controlled conditions, measuring the distances travelled through gel by said nucleic acids, and marking permanent indications at same distances on said artificial rectangular shaped travel -distance scale of length equivalent to said gel to denote the start of gel run, the stop of gel run, and the specific base pair lengths.

One more aspect of the disclosure is an artificial permanent ladder for identification and analysis of proteins using gel electrophoresis comprising a rectangular shaped travel-distance scale having a top portion having marking indicating the start of gel run, a bottom portion having marking indicating the stop of gel run and a middle portion marked with indications at various distances which corresponds to molecular weights of specific proteins. The relation between the indications and the molecular weights are derived after running more than one gel electrophoresis experiments for proteins of known molecular weights under controlled conditions, measuring the distances travelled through gel by said proteins, and marking permanent indications at same distances on said artificial rectangular shaped travel-distance scale of length equivalent to said gel to denote the start of gel run, the stop of gel run, and the specific molecular weights. These permanent standard ladders are used in place of standard ladders in gels during every electrophoretic run of a nucleic acid samples or protein samples.

Yet one more aspect of the disclosure is a method of running simultaneous electrophoresis for nucleic acids or proteins in a single electrophoresis apparatus of the present disclosure in gels of different concentrations by arranging an electrophoresis apparatus having the following components

- buffer tray to hold the electrophoretic buffer in said apparatus; either glass slabs with side spacers making an inner cavity or a gel casting tray having a space for setting and holding of the electrophoretic gel in said apparatus; an anode and a cathode for electric supply; a rectangular planar body (600) having alternating portions one being a basal portion tooth-spacer (603 B) with thickness and width appropriate to accommodate a detachable tooth (604) or a separator (606) and having means of attachment for attaching detachable teeth

(604), and another being an elevated portion tooth-holder (603 A), and

- two side edges for resting said rectangular planar bodyon saidside spacers or said gel casting tray; one or more separators (606) for compartmentalizing said inner cavity of the glass slabs or said space of gel casting tray for setting and holding multiple gels within the said apparatus and attaching detachable teeth (604) to said rectangular planar body (600) with attaching one or more said separators(606) to said rectangular planar body(606), resting said rectangular planar body on said side spacers and placing the said separators at required positions within inner cavity bound by said glass slabs and removing the rectangular planar body with attached said detachable teeth or attaching detachable teeth (604) to said rectangular planar body (600) placed within said gel casting tray and aligning the said separators placed within the cavity of gel casting tray with said empty tooth-spacers on the said rectangular planar body and removing the rectangular planar body with attached said detachable teeth, followed by adding gels of varying concentrations in different compartments created by compartmentalizing said inner cavity of the glass slabs or said space of gel casting tray and resting the said rectangular planar body (600) with attached said detachable teeth said side spacers or said gel casting tray within dipping of attached teeth in the gels multiple compartments in said inner cavity of the glass slabs or said space of gel casting tray and allowing said gels of varying concentrations to set in said compartments with creation of wells around detachable teeth and thereafter removing said rectangular planar body (600) along with the detachable teeth attached to it from the electrophoretic apparatus, followed by introducing the nucleic acid or protein in the wells created in gels around detachable teeth and starting the electric supply for running simultaneous electrophoresis of nucleic acid or protein ingels of different concentrationsin said single electrophoresis apparatus and analysing the obtained bands in various gels by using suitable detection techniques.

For running PAGE , the front and and back glass slabs are assembled with two regular spacers on left and right side, by fixing the whole assembly with the help of adhesive tape. The bottom of the glass slabs are attached to buffer tray usingagarose gel. In the next step, required number of detachable teeth of present disclosure are placed on the required toothspacers of the comb body of the present disclosure, leaving other tooth-spacers empty for holding of the separators as per the requirement of comparments within the cavity. The teeth are aligned to required length and fixed properly with protruding circular slots. The PAGE separators of the present disclosure are then placed on empty tooth- spacers within the comb body in such a manner than when the whole comb is placed on the PAGE assembly, the separators will cover the entire length of the cavity thereby effectively resulting into compartmentailzation of the cavity. The comb is then placed on the PAGE assembly. For protein separation, required amount of resolving gel solution of specific concentrationis introduced within one of the compartment to cover almost 70% of the available length. Similarly resolving gel solution of another concentration is introduced in the other compartment to cover almost 70% of the available length. Then the stacking gel solutions of required concentrations are introduced, each in its corresponding cavity. The stacking gel solutions are allowed to polymerize around the comb teeth with creation of wells in place of the teeth. For nucleic acid separation, per compartment only one gel solution is introducedand the gel solutions in all the compartments are allowed to polymerise around the comb teeth for creationof wells [Fig 7(a)]. The comb along with teeth is removed once the gelsare firmly polymerised. The separator is kept within the assembly. The assembly is filled with the running buffer and isnow ready for sample loading and electrophoresis. The samples mixed with tracking dye are introduced in the wells of each comparment. The assembly is then filled with the gel buffer . The assembly is supplied with electric current and the seperations is allowed to carry out. [Fig 7(b)].

Once the electrophoretic run is over( the end of the run based on the loading dye migration is also marked in the front of the glass) and the gel is removed from the assembly and stained with a suitable stain base upon the nature of sample(nucleic acid or protein). The bands are then visualized under UV or visible light dependeing upon the nature of stain used. A 'gelcard' containing band markings for peptides of different molecular weights or nucleic acid of different base pair length under required concentration of polyacrylamide is used in place of 'standard peptide ladder' for comparison and determination of molecular weight of peptides for base pair length of nucleic acids in samples.

For running AGE, the open ends of the casting tray are sealed with cellotape and the tray is tested for leakage by pouring water into it. Required number of teeth are aligned and fixed on the protruding circular slots on the comb within toothholders except for those slots which are corresponding to the separators for cavity compartmentalization. The comb is then placed on the casting tray and position of separators is marked. The separators of the present disclosure are the placed in casting tray at a position just below the comb which is corresponding to the tooth-slots, where teeth are missing. Following to this, the agarose solutions of required concentrations are poured, one into each compartment created by separators. [Fig. 8] The gels are then allowed to solidify around comb teeth to get wells within the gel. After settling of the gel, the comb is removed and the casting tray is placed within electrophoresis chamber. The electrophoretic buffer is them poured carefully in the chamber, taking care that all the wells are filled with the buffer. Then samples mixed with loading dye and are loaded in the wells in each compartment. The electrophoresis is carried out under influence of electric current between cathode and anode. [Fig 9]. Once the electrophoretic run is over, the gel is removed carefully from casting tray and is stained with a suitable stain. The gel is then visulaization either under UV light or in visible light depeneding upon the nature of the stain. A 'gelcard' containing band markings nucleic acid of different base pair length under required concentration of agarose is used in place of 'standard peptide ladder for comparison and determination base pair length of nucleic acids in samples.

The present disclosure provides a technical advancement and has economic significance in the sense that it allows running of samples in multiple gels at one go and thus facilitates better results are all experimental conditions are uniform for multiple gels of different concentrations and hence helps a researcher to make a choice of gel type and its concentration in a better manner are experimental conditions are not varying as in case of multiple runs. The economic significance of the disclosure is that it reduces the amounts of reagents which are otherwise used during multiple runs and it also circumvents the need of running a standard ladder of nucleic acid or protein during every run of electrophoresis by introducing the permanent standard ladder 'gelcard' thereby reducing the cost.

EXAMPLES

Case Study I

A case study was carried out on PAGE assembly of MICROKIN company to design the comb, detachable teeth and separator for compartmentailization. Standard DNA ladder of 100 bp was run on this assembly to prepare a 'gelcard'.

Step I - Preparation of comb with detachable teeth and separator a. Based upon the measurements of the MICROKIN PAGE assembly, following dimensions& specifications of the comb body, detachable teeth and separator for compartmentlization were calculated -

Comb Body -

Dimensions - 11.2 cm X 4.5 cm X 0.2cm First cut from top on both the sides (to rest the comb body on side separators) - 3 cm

Depth of First Cut - 2.1 cm from each side

Second cut from top on both the sides(to fit the width of body within cavity width) - 4 cm

Depth of Second cut - 2.6 cm from each side

Total height of the comb body - 4.5 cm

No of tooth-holders on the comb body - 10

Dimensions of a tooth-holder - 0.3 cm X 4.5 cm X 0.2 cm Distance between two tooth-holders - 0.4 cm

No of tooth-spacers - 9 (One tooth spacer capable of holding one detachable tooth between two tooth-holders)

No. of pin-holders on each tooth- spacer -3

Distance of of first pinholder from top - 1 cm Distance between any two pinholders - 0.2 cm

Detachable tooth -

Dimensions - 7.5 cm X 0.4 cm X 0.2 cm

No. of sets of means of attachment - 2

No. of means of attachment in each set - 3 Distance of first means of attachment of first set from top - 3 cm

Distance of first means of attachment of second set from top - 5.5 cm

Distance between any two means of attachments of a set - 0.2 cm Separator for compartmentalization - Dimensions - 12 cm X 0.4 cm X 0.2 cm

Step II - Preparation of comb body, detachable teeth, separators and spacers

All parts were prepared using high - grade acrylic which is neutral to all the chemicals used in PAGE. Comb body was cut out from a piece of 15 cm X 5 cm rectangular piece. Distance of First cut and second cut from the top and both left and right side were marked and cuts were made in the piece. All cuts were made using a Laser Cutting Tool. The locations of all means of attachments were marked on the comb body by calculating distances from top and left. The holes were made in the comb body to carve the means of attachment with a laser driller. A horizontal rester was attached to the comb body on the side of toothholder just above the first cut. The rester was of the dimensions 10 cm X 0.5 cm X 0.2 cm and approximately 'C shaped to ensure resting of the comb on top of spacers.

10 tooth-holders of above mentioned dimensions were cut out from a 5 cm X 5 cm rectangular piece using Laser Cutting Tool. 9 detachable teeth of above mentioned dimensions were cut from a 10 cm X 5 cm rectangular piece using a Laser Cutting Tool. Locations of means of attachment of both the sets were marked on each tooth and holes were made using a laser drill.

The separator of specified dimensions was cut from an acrylic piece of suitable size.

2 spacers of the dimesions 11cm X 2 cm X 0.2cm were cut out of a larger acrylic sheet. A U shaped cut was made in the center of each spacer of the dimensions 10cm X 0.5 cm from the bottom. A rectangular area of the dimensions 4cm X 0.5 cm was cut from the top right edge of the left hand spacer to create a notch in it. A marking was made 0.5 cm below this notch. In a similar manner a rectangular are of dimensions 4cm X 0.5 cm was cut from the top left edge of the right hand spacer to create a notch in it and marking is made 0.5 cm below this notch. A second marking was made 6.0 cm below this notch. All cuttings were made with Laser Cutting Tool

Step III - PAGE Apparatus assembly

Four detachable teeth were attached to the comb body from left by aligning all three means of attachment of second set of one tooth with all three means of attachment on one tooth-spacer and fixing them with three attachment tools. In a similar fashion, four more deachable teeth were attached to the comb body from right. No detachable tooth was attached in the middle tooth-spacer.

The front and back glass slabs of MICROKIN PAGE apparatus were assembled with side spacers between them on left and right side with the help of the screws of the apparatus to create a cavity between them. The comb with detachable teeth was then placed on the top of the side separators. The separator for compartmentailization was carefully introduced in the cavity by sliding it through the middle empty tooth-spacer from the top. Bottom of the glass slides resting on the reservoirwas sealed with 8% agarose gel solution.

Step IV - Gel Preparation, sample loading and electrophoretic run -

12 ml of each of Polyacrylamide gels of the concentrations 8% and 12% are prepared from the reagents given in Table 1

Table 1 : - Reagents quanity for preparation polyacrylamide gels of different concentration

*30% Acrylamide stock solution is comprised of 29:1 (% w/v) acrylamide:bisacrylamide combination

TBE - Tris- Borate- EDTA buffer; APS- Ammonium Persulfate;

TEMED - Tetramethylethylenediamine Required quantities of all reagents were mixed well except for TEMED and mixed well. TEMED was added to one of the mixtures at a time, mixed quickly and immediately introduced in the target compartment in the cavity upto notch level of spacers (6.5 cm from botton) between glass slabs of PAGE assembly. Both the gels were allowed to polymerize and then the comb with attached teeth was removed, except for the compartmentalization separator.

Wells in both the gels were then flushed with lx TBE buffer. Markings were made on the front glass slab to mark the wells, lx TBE buffer was introduced gently in the cavity above the wells in both compartment to a level little bit above the wells. IX TBE buffer was also introduced in the reservoir. 10 microliters of sample mixed with loading dye was then carefully introduced into three wells in each gel. Similarly standard ladder of 100 bp was also introduced into one well in each gel. Anode and cathode was attached to a battery and electrophoretic run was carried out at a voltage of 7V/cm. As the loading dye neared the end of the gel upto the second marking on spacers, electric current was discountinued and electrophoresis was stoped. The glass slabs along with seprators was removed from the assembly and the gels were carefully removed from the slabs and placed in staining tray. The gels were stained with ethidium bromide for about 30 minutes and then thourougly washed with distilled water. The bands were then visualized under UV light of GelDoc.

Step V - The gels prepration, sample loading and electrophoretic run as described in step IV was repeated three times. The distance travelled by each band of standard ladder in each gel of specific concentration was measured in all three repeats and a mean distance travelled by each band was calculated and a 'gelcard' was prepared for 100 bp stanadard ladder for 8% Polyacrylamide concentration which showed a better resolution compared to 12%

Step VI - The gel preparation, sample laoding and electrophoretic run was repeated once more except for running of 'standard ladder' for 8% polyacrylamide gel. The sample bands were visualized with 'gelcard' in the place of standard ladder Results

Polyacrylamide solution of 8% concentration sucessfully geled in one of the compartments without entering the other one. Similarly polyacrylamide solution of 12% concentration sucessfully geled in the other compartment without entering the first one. Thus compartmentalization of the cavity between front glass slab & back glass slab with polymerization of gels of two different concentrations without intermixing was sucessfully achieved using the separator of present disclosure. [Fig 10 ]

Three bands in each sample and 10 bands in standard ladded seprarated in each gel. The distances travelled by each standard and sample are in good agreement in triplicates of 8% polyacrylamide gel [Fig 1 1]. Distance travelled by each standard and sample are not in so good agreement in triplicates of 12 % polyacrylamide [Fig 12], the reason being higher concentration of polyacrylamide. The resolution of standard ladder is in better in case of 8% polyacrylamide gelcompared to 12% polyacrylamide gel[Table 2]

8 300 bp 30.6 29.4 30.5 30.2 18.3 19.8 20.3 19.5

9 200 bp 37.2 35.9 37.0 36.7 20.3 21.3 22.9 21.5

10 100 bp 46.0 45.0 46.0 45.7 23.4 24.4 25.4 24.4

11 SB 1 23.4 22.5 23.2 23.0 14.2 15.2 16.3 15.2

12 SB2 36.3 35.0 36.2 35.8 19.3 20.3 21.3 20.3

13 SB 3 38.2 36.8 38.0 37.7 20.3 21.3 22.3 21.3

Table 2 - Distance Travelled by Standard and Sample Bands

*SB1- Sample band 1 - 475 bp, SB2- Sample band 2- 214 bp, SB3- Sample band 3- 178 bp

For the polyacryalmide gel of 8%concentration, difference between two consecutive bands of standard ladderis in good agreement with a variation of maximum 0.2 mm. The distance of sample band lof legnth 475 bp the from nearest standard band of 500 bp is in the range of 0.4 to 0.5 mm. Similarly the distances of sample band2 (214 bp) sample band 3(178 bp) from the nearest standard band of 200 bp is range are in the range of 0.8 to 0.9 mm and 0.9 to 1.0 mm respectively. For the polyacrylamide gel of 12% concentration, difference between two consecutive bands of standard ladder not in good agreement with a maximum of variation in the range of 1 mm. [Table 3]

5 600 bp - 500 bp 3 3 3 3.0 3.0 3.0

6 500 bp - 400 bp 2.8 2.8 2.8 1.0 2.0 2.0

7 400 bp - 300 bp 4.8 4.8 4.8 3.0 2.5 2.0

8 300 bp -200 bp 6.6 6.6 6.6 2.0 1.5 2.5

9 200 bp -100 bp 8.8 8.8 8.8 3.0 3.0 2.5

10 SBl - 500 bp 0.4 0.4 0.4 0.0 0.0 0.0

11 SB2 - 200 bp 0.9 0.9 0.9 -1.0 -1.0 -1.5

12 SB3 - 300 bp 1 1 1 0.0 1.0 -0.5

Table 3 - Distance between consecutive bands of standard ladder and sample bands& nearest standard band

Distance travelled by samples in 8% polyacrylamide gel was in good agreement with the 'gelcard' of 100 bp ladder[Fig 15 (a) & Fig 15 (b)].

Conclusion:- In conclusion, the comb with detachable teeth, separators and spacers were sucessfully used for compartmentalization of PAGE gel cavity and polymerization of polyacrylamide solutions of two different concentrations, one in each compartment without intermixing and creation of wells within both the gels by detachable teeth of the comb. It is also concluded that the whole assembly was sucessfully used for electrophoretic run of samples and standards in both gels simultaenously. A 'gelcard' prepared for 100 bp ladder for 8% polyacrylamide gel which was used for confirming the base pair lengths of samples in accurate manner.

Case Study II

A case study was carried out on AGE assembly of SUB SLEEK company to design the comb, detachable teeth and separator for compartmentailization.

Step I - Preparation of comb with detachable teeth and separator a. Based upon the measurements of the SUB SLEEK AGE assembly, following dimensions & specifications of the comb body, detachable teeth and separator for compartmentlization were calculated -

Comb Body - Dimensions - 10 cm X 4 cm X 0.2cm

Cut from top on both the sides (to rest the comb body on gel casting tray) - 1.5 cm

Depth of First Cut - 1.5 cm from each side Total height of the comb body - 4 cm No of tooth-holders on the comb body - 8

Dimensions of a tooth-holder - 0.3 cm X 4 cm X 0.2 cm Distance between two tooth-holders - 0.4 cm

No of tooth-spacers - 9 (One tooth spacer capable of holding one detachable tooth between two tooth-holders as well as bound by one tooth holder on left and right extremes)

No. of means of attachment on each tooth- spacer -3

Distance of of first means of attachment from top - 1 cm

Distance between any two means of attachment - 0.2 cm

Detachable tooth - Dimensions - 7.5 cm X 0.4 cm X 0.2 cm

No of sets of means of attachment - 2

No. of means of attachment in each set - 3

Distance of first means of first set from top - 3 cm

Distance of first means of second set from top - 5.5 cm Distance between any two means of attachments of a set - 0.2 cm Separator for compartmentalization -

Dimensions - 10 cm X 0.4 cm X 1 cm

Step II - Preparation of comb body, detachable teeth, separators and spacers All parts were prepared using high - grade acrylic which is neutral to all the chemicals used in AGE. Comb body was cut out from a piece of 15 cm X 5 cm rectangular piece. Distance of cut from the top and both left and right side were marked and cuts were made in the piece. All cuts were made using a Laser Cutting Tool. The locations of all means of attachments were marked on the comb body by calculating distances from top and left. The holes were made in the comb body to carve the means of attachments with a laser driller. A horizontal rester was attached to the comb body on the side of toothholder just above the first cut. The rester was of the dimensions 10.5 cm X 0.5 cm X 0.2 cm and approximately 'C shaped to ensure resting of the comb on the gel casting tray 8 tooth-holders of above mentioned dimensions were cut out from a 5 cm X 5 cm rectangular piece using Laser Cutting Tool.

9 detachable teeth of above mentioned dimensions were cut from a 10 cm X 5 cm rectangular piece using a Laser Cutting Tool. Locations of holes of means of attachments both the sets were marked on each tooth and holes were made using a laser drill.

The separator of specified dimensions was cut from an acrylic piece of suitable size.

Step III - AGE Apparatus assembly

Four detachable teeth were attached to the comb body from left, leaving the first slot empty, by aligning first means of attachment of second of one tooth with last means of attachment on one tooth-spacer and fixing them with attachment tool. In a similar fashion, three more deachable teeth were attached to the comb body from right. No detachable tooth was attached in the fifth tooth-sapcer.

Comb attached with the detachable teeth was placed on the gel casting tray at a distance from 1 cm from one of the cellotape sealed end. Circled cellotape was attached to the separator to ensure that it will stick to the separator as well as casting tray. The separator was firmly stuck within the cavity of casting tray by carefully placing it below the fifth toothspacer of the comb. The comb was then removed and the open ends of the were sealed with the cellotape taking care that the tape also stuck to ends of the separator. The gel tray was check for leakage by pouring water in it. There was no leakage. The water was removed and the surface was wiped with tissue paper.

Step IV - Gel Preparation, sample loading and electrophoretic run -

30 ml of each of Agarose gels of the concentrations 0.8% and 1.7% are prepared from the reagents given in Table 1

Table4 : - Reagents quanity for preparation agarose gels of different concentration

5X TAE Buffer - 24.2gm Tris base+5.71ml acetic acid &+10ml 0.5M EDTA +

Sufficient distilled water =100 ml buffer

EtBr - Ethidium bromide - 10 mg/ ml in distilled water

Required quanty of water and 5X TAE buffer was mixed, then agarose was added to it and the solution was heated to dissolve the agarose. Ethidum bromide solution was then added to the agarose solution and mixed.

0.8 % agarose solution was added to one of the compartment within gel casting tray to a level just below the separator level. Similarly 1.7% agarose solution was added to the second compartment within gel casting tray to a level just below the separator level. The comb was then placed on the gel casting tray by immersing the teeth into gel and allowed to solidfy[Fig 13].

On solidifications little bit IX TAE was poured on both gels and the comb was carefully removed from the gels. The wells were created in the place of the teeth of the comb.

Conclusion :- In conclusion, the comb with detachable teeth, separators were sucessfully used for compartmentalization of cavity of AGE gel casting tray and geling of agaorse solutions of two different concentrations, one in each compartment without intermixing and creation of wells in each gel by detachable teeth of the comb.

Claims

gel electrophoresis apparatus having modified comb comprising - a buffer tray to hold the electrophoretic buffer in said apparatus;

either glass slabs with side spacers making an inner cavity or a gel casting tray having a space for setting and holding of the electrophoretic gel in said apparatus;

an anode and a cathode for electric supply;

a modified comb to accommodate detachable teeth and separators within it, said modified comb essentially consisting of

a rectangular planar body(600)having alternating portions one beinga basal portion tooth-spacer (603 B) having means of attachment for attaching detachable teeth (604)and another being an elevated portion tooth-holder (603 A),

two side edges for resting said modified comb on said side spacers or said gel casting tray,

detachable teeth (604) having means of attachment for getting attached with said tooth-spacer portions,

an attachment tool (607) for attaching said detachable teeth

(604) with said tooth-spacer (603 B),and

one or more separators (606) for compartmentalizing said inner cavity of the glass slabs or said space of gel casting tray for setting and holding multiple gels within the said apparatus, wherein the tooth-spacer is the basal portion of thickness and width appropriate to accommodate a tooth or a separator and the tooth- holder is the elevated portion of thickness and width appropriate for supporting and placement of tooth or separator in adjacent tooth- spacer.

2. The gel electrophoresis apparatus having modified comb as claimed in claim

1, wherein the detachable teeth (604) are of thickness appropriate to match the level of elevation of the tooth-holder, of width appropriate to fit into the tooth-spacer firmly and of length enough to dip in the gel set in the inner cavity of the glass slabs or in space of gel casting tray.

3. The gel electrophoresis apparatus having modified comb as claimed in claim

1, wherein the separator (606) is of thickness, width and height appropriate to compartmentalize the inner cavity of the glass slabs or the space of gel casting tray.

4. The gel electrophoresis apparatus having modified comb as claimed in claim

1, wherein the attachment tool (607) is adhesive band.

5. The gel electrophoresis apparatus having modified comb as claimed in claim

1, wherein the means of attachment of detachable teeth (604) and means of attachment of a tooth-spacer are (603 B) are holes having equal dimeters, and the detachable teeth (604) are attached to tooth-spacers (603 B) using the attachment tool (607) that percolate through said holes.

6. The gel electrophoresis apparatus having modified comb as claimed in claim

5, wherein the attachment tool (607)is pins.

7. The gel electrophoresis apparatus having modified comb as claimed in claim

1, wherein the means of attachment of detachable teeth (604) and means of attachment of a tooth-spacer (603 B) are glueable portions, and the detachable teeth (604) are attached to tooth-spacers (603 B) using an adhesive.

8. The gel electrophoresis apparatus having modified comb as claimed in claim

1, wherein the gel electrophoresis refers to a polyacrylamide gel electrophoresis or an agarose gel electrophoresis of nucleic acid samples, or a SDS polyacrylamide gel electrophoresis of proteins.

9. The gel electrophoresis apparatus having modified comb as claimed in claims

1 and 8, wherein said rectangular planar body, side edges, detachable teeth, attachment tool and one or more separators are made of a material inert to all reagents of polyacrylamide gel electrophoresis and agarose gel electrophoresis.

10. An artificial permanent ladder for identification and analysis of nucleic acids using gel electrophoresis comprising a rectangular shaped travel-distance scale having

a top portion having marking indicating the start of gel run;

a bottom portion having marking indicating the stop of gel run; and a middle portion marked with indications at various distances which corresponds to base pair lengths of specific nucleic acids,

wherein said correspondence relation between the indications and the base pair lengths are derived after running more than one gel electrophoresis experiments for nucleic acids of known base pair lengths under controlled conditions, measuring the distances travelled through gel by said nucleic acids, and marking permanent indications at same distances on said artificial rectangular shaped travel-distance scale of length equivalent to said gel to denote the start of gel run, the stop of gel run, and the specific base pair lengths.

11. An artificial permanent ladder for identification and analysis of proteins using gel electrophoresis comprising a rectangular shaped travel-distance scale having

a top portion having marking indicating the start of gel run;

a bottom portion having marking indicating the stop of gel run; and a middle portion marked with indications at various distances which corresponds to molecular weights of specific proteins,

wherein said correspondence relation between the indications and the molecular weights are derived after running more than one gel electrophoresis experiments for proteins of known molecular weights under controlled conditions, measuring the distances travelled through gel by said proteins, and marking permanent indications at same distances on said artificial rectangular shaped travel-distance scale of length equivalent to said gel to denote the start of gel run, the stop of gel run, and the specific molecular weights.

12. A method of running simultaneous electrophoresis for nucleic acids or proteins in a single electrophoresis apparatus in gels of different concentrations comprising

a) arranging an electrophoresis apparatus having following components buffer tray to hold the electrophoretic buffer in said apparatus; either glass slabs with side spacers making an inner cavity or a gel casting tray having a space for setting and holding of the electrophoretic gel in said apparatus; an anode and a cathode for electric supply; a rectangular planar body (600) having alternating portions one being a basal portion tooth-spacer (603 B) with thickness and width appropriate to accommodate a detachable tooth (604) or a separator (606) and having means of attachment for attaching detachable teeth (604), and another being an elevated portion tooth-holder (603 A), and two side edges for resting said rectangular planar bodyon saidside spacers or said gel casting tray; one or more separators (606) for compartmentalizing said inner cavity of the glass slabs or said space of gel casting tray for setting and holding multiple gels within the said apparatus; and b) attaching detachable teeth (604) to said rectangular planar body (600) and attaching one or more said separators(606) to said rectangular planar body(606), resting said rectangular planar body on said side spacers and placing the said separators at required positions within inner cavity bound by said glass slabs and removing the rectangular planar body with attached said detachable teeth or attaching detachable teeth (604) to said rectangular planar body (600) placed within said gel casting tray and aligning the said separators placed within the cavity of gel casting tray with said empty tooth- spacers on the said rectangular planar body and removing the rectangular planar body with attached said detachable teeth; c) adding gels of varying concentrations in different compartments created by compartmentalizing said inner cavity of the glass slabs or said space of gel casting tray; d) resting the said rectangular planar body (600) with attached said detachable teeth said side spacers or said gel casting tray within dipping of attached teeth in the gels multiple compartments in said inner cavity of the glass slabs or said space of gel casting tray; e) allowing said gels of varying concentrations to set in said compartments with creation of wells around detachable teeth and thereafter removing said rectangular planar body (600) along with the detachable teeth attached to it from the electrophoretic apparatus; f) introducing the nucleic acid or protein in the wells created in gels around detachable teeth; g) starting the electric supply for running simultaneous electrophoresis of nucleic acid or protein in gels of different concentrations in said single electrophoresis apparatus; and h) analysing the obtained bands in various gels by using suitable detection techniques.