KR20160142530A - Composition of Anti-freezing agents - Google Patents

Abstract

The present invention relates to an anti-freezing agent composition capable of stably storing nucleic acids, proteins and the like. More specifically, according to the present invention, synthesized oligonucleotides, genomic DNA, PCR (RT/PCR) master mixture, restrictive enzymes and the like can be dissolved in a solution containing the anti-freezing agent which does not freeze even at temperatures of -20C or lower, and thereby can be stored conveniently and stably, while the inconvenience of performing repeated freezing/thawing cycles for each use and the problem of decreased performance can be minimized.

Description

Composition of Anti-freezing agents [

The present invention relates to a cryoprotectant composition for a biomaterial, and more particularly, to a cryoprotectant composition capable of storing a bio-material such as a nucleic acid and a protein for a long period of time without lowering the activity.

The present invention relates to a cryoprotectant capable of stably storing a synthetic mixture (oligonucleotide), a genomic DNA, RNA, and a PCR (RT / PCR) master mixture, which are widely used for PCR, cloning, hybridization and the like Freezing agents (AFA) that do not freeze at temperatures below -20 ° C in place of aqueous solutions such as distilled water or tris buffer solutions, which are commonly used solvents, genome DNA, and PCR (RT / PCR) master mixture by minimizing the problems such as time consuming and freezing / thawing process, .

Generally, biomaterials such as proteins and DNA are not stored in aqueous solution. Therefore, they are stored in an ultra-low temperature of -20 ° C or lower rather than room temperature in order to maintain their activity. They are thawed if necessary, . However, such repetitive freezing and thawing causes the protein's normal secondary structure to change and the protein to lose its activity (Jameel F., Bogner R., Mauri F. and Kalonia D., J. Pharm Pharmacol., 49 ), 472-477, 1997, Nardid O., Dyubko T. and Repina S., Cryobiology, 34 (2), 107-113, 1997). When proteins are stored for a long period of time, several methods of drying are used to stabilize them. Among these methods, in the case of storing an enzyme such as a restriction enzyme or a DNA polymerase by a freeze drying method, And the activity thereof is lost in the thawing process. It must also be completely frozen at various temperatures and rates depending on the type of material being lyophilized. The freeze-drying device required here is expensive and the freeze-drying process is relatively slow. Such economic factors and hassles of operation make it difficult to use (see Korean Patent Registration No. 10-0857889).

The synthetic genes used for PCR are generally used by dissolving in TE buffer or distilled water and stored frozen below -20 ℃ for long-term storage stability. Therefore, not only the inconvenience of melting and using each time of use, but also the repetitive freezing and thawing cause a decrease in the activity of the synthetic gene, so it is necessary to use it in small batches.

In the case of genomic DNA, for long-term storage, it is stably stored for 1-2 years at a temperature of 0-4 ° C in an aqueous solution containing lyophilized or 70% ethanol. However, in the case of the genomic DNA, it is generally stored at 4 ° C in order to prevent the decrease in activity due to repeated freezing / thawing. In this case, the DNA can be stably stored for up to 6 months. The stability is deteriorated drastically.

For various enzymes such as restriction enzymes, DNA polymerase and reverse transcriptase, which are most frequently used in molecular biology experiments, 50% glycerol buffer solution is usually used. The added glycerol lowers the freezing point of the enzyme to freeze the enzyme (See U.S. Patent No. 5,250,429). However, due to the high viscosity, the accuracy of dispensing at 1-2 μL is reduced. In the case of restriction enzyme, when the concentration of glycerol is 5% or more of the total reaction solution, star activity (star reaction) There is a limit to the amount of restriction enzyme used. Further, in order to minimize the inconvenience of the reaction liquid preparation and minimize the experimental error, a master mixture in which a buffer, dNTP, primer and the like are mixed is often prepared and stored, but there is a limit to stable storage.

Korean Patent Publication No. 2002-0013521 discloses a composition for preserving an infectious recombinant adenovirus, which comprises a buffer solution supplemented with glycerol and capable of maintaining a medium pH of 8.0 to 9.6. A liquid or frozen composition is disclosed.

Korean Patent Laid-Open Publication No. 2008-0087148 discloses a low-temperature protection composition comprising a nanostructure, a liquid, and at least one low-temperature protection agent comprising nanometer-sized core material surrounded by liquid regular liquid molecules.

Korean Unexamined Patent Application Publication No. 2005-0030416 discloses a method for cryopreserving a shellfish larvae, comprising the steps of: placing the shellfish larvae in an artificial seawater diluted with a cryoprotectant to absorb the cryoprotectant; Placing the floating larvae at room temperature for an equilibration time, and cooling the coarse floating larvae after the equilibration time by using a freezer.

The present invention relates to a composition and a method for cryopreservation at low temperatures. In addition to glycerol, a method of adding a buffer for maintaining pH at alkaline, adding a nanostructure, or adding artificial seawater is used, And the effect of the present invention in which freezing is prevented even at -30 캜 or lower.

Accordingly, it is an object of the present invention to provide a cryoprotectant composition that can stably store biomaterials such as nucleic acids and proteins even at a low temperature below freezing and does not suffer from loss of activity due to freezing / thawing.

In order to solve such a problem, it is necessary to develop a composition capable of stably preserving the biomaterial through identification of kinds and composition ratios of the components contained in the composition for cryopreservation. Under these circumstances, the present invention provides an anti-freezing agent (anti-freezing agent) that does not freeze at a temperature below -20 ° C instead of an aqueous solution in order to minimize troubles such as usage and reduction in activity, , AFA), and confirmed the proper composition ratio between the components and the components that do not inhibit the activity or stability of the biomaterial, and completed the present invention.

The present invention relates to a method of freezing a biomaterial containing at least one of proteins or nucleic acids, which comprises a combination of at least one of ethanol or methanol and glycerol, at a temperature of 0 ° C or lower and not higher than -40 ° C, ≪ / RTI >

The present invention also relates to the combination of at least one of 10-40% (v / v) ethanol or 10-30% (v / v) methanol and 10-40% (v / v) glycerol And a cryoprotectant composition.

Also, the present invention relates to a cryoprotectant composition, wherein the biomaterial is at least one selected from the group consisting of a polymerase, a restriction enzyme, a reverse transcriptase, DNA, RNA, oligonucleotide and dNTP.

In addition, the present invention relates to a cryoprotectant composition comprising at least one of 20-40% (v / v) ethanol or 20-30% (v / v) methanol and 20-40% (v / v) glycerol The present invention also relates to a cryoprotectant composition which can be stably stored at a temperature not higher than 0 ° C and not lower than -40 ° C even when it is diluted to 1/2 at the time of work.

In addition, the present invention includes a combination of at least one of 20-40% (v / v) ethanol or 20-30% (v / v) methanol and 20-40% (v / v) glycerol, A cryoprotectant composition capable of stably storing freezing at a temperature not higher than 0 ° C and not lower than -40 ° C even when diluted to 1/2 at the time of operation and one or more biomaterials of protein and nucleic acid at a volume ratio of 1: And a method of using the same.

The cryoprotectant of the present invention

1) It can be prepared at a concentration more than twice the working concentration,

2) Biomaterials such as oligonucleotides, genomic DNA, RNA, PCR (RT / PCR) master mixtures and restriction enzymes in working concentrations are not frozen even when stored at -20 ° C,

3) Even after storage at -20 ° C for a long period of time, there is no influence on the biological activity of the sample.

4) In the case of PCR reaction, up to 30% (up to 6μl in 20μl reaction solution) of the reaction solution to be used does not affect the enzyme reaction,

5) The reduction of star activity in restriction enzymes made it easier to use more restriction enzyme than 50% glycerol.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a graph showing freezing points of the cryoprotectant according to the present invention. FIG.
FIG. 2 is an agarose gel photograph showing the effect of the cryoprotectant of the present invention on PCR using several pairs of primers.
3 is an agarose gel photograph showing the maximum amount of the cryoprotectant of the present invention which can be used for PCR.
Figure 4 is a photograph of agarose gel comparing the effect of the cryoprotectant of the present invention on the detection sensitivity of PCR.
5 is a photograph of agarose gel comparing the stability of deoxyoligonucleotides dissolved in the cryoprotectant of the present invention.
6 is an agarose gel photograph comparing the stability of the genomic DNA dissolved in the cryoprotectant of the present invention.
7 is an agarose gel photograph comparing the stability of a PCR master mixture comprising the cryoprotectant of the present invention.
Figure 8 is a photograph of agarose gel comparing the stability of the reverse transcriptase master mixture comprising the cryoprotectant of the present invention.
Figure 9 is a photograph of agarose gel comparing the freeze / thaw effect of the reverse transcriptase master mixture dissolved in distilled water.
10 is an agarose gel photograph comparing the stability of RNA dissolved in the cryoprotectant of the present invention.
11 is a photograph of agarose gel comparing freezing / thawing effects of RNA dissolved in distilled water.
12 is a photograph of agarose gel comparing the stability of the restriction enzyme stored in the cryoprotectant of the present invention.
13 is a photograph of agarose gel comparing the star activity of the restriction enzyme stored in the cryoprotectant of the present invention.
FIG. 14 shows the stability and reactivity of the deoxyoligonucleotide and the PCR master mixture dissolved in the cryoprotectant of the present invention measured by real-time PCR.

Hereinafter, the configuration of the present invention will be described in more detail with reference to examples. It is to be understood by those skilled in the art that these embodiments are merely illustrative of the present invention and that the scope of the present invention is not limited to these embodiments.

Example  1: Preparation of antifreeze solution

Generally, the 50% glycerol solution does not freeze at -20 ° C. However, an aqueous solution containing 50% glycerol is not easily pipetted at 1 to 2 μl due to the viscosity of glycerol.

Generally, 50% glycerol solution has several disadvantages because it can dilute the sample to the desired concentration so that the storage solution can be prepared at a concentration of at least twice the working concentration in the laboratory.

Therefore, in the present invention, at least one of ethanol or methanol is added to minimize the concentration of glycerol and the freezing point of the solution, and a predetermined amount of ethanol and / or methanol is added to the glycerol concentration of 20% and 10% 30 < 0 > C and -35 < 0 > C for 24 hours. As a result, the freezing point of each solution was as shown in Fig.

As shown in FIG. 1, when the solution was not frozen at -30 ° C in the case of adding 14% of ethanol to 20% of glycerol or 11% of methanol to 20% of glycerol and when 20% of ethanol and 14% of methanol were added to 10% of glycerol, The solution did not freeze even at 30 ° C. Therefore, the appropriate amount of methanol and ethanol can lower the concentration of glycerol to 10-20%, which makes it possible to add 24-40% ethanol to 20-40% glycerol, which is twice the working concentration, or 22-40% ethanol, A new cryoprotectant solution having a methanol concentration of 28% can be prepared.

Example  2: Cryoprotectant solution PCR Effect on

To investigate the effect of 20-40% glycerol + 24-40% ethanol or 20-40% glycerol + 22-28% methanol solution prepared in Example 1 on the reactivity of PCR (Polymerase Chain Reaction) The reactivity of PCR was determined by adding a fixed amount of aqueous solution containing 20% of glycerol to 10, 11, 12 and 13% of methanol and 20% of glycerol and 13, 14, 15 and 16% of ethanol, 2 of AD, and did not affect the PCR reaction at all. Lane 1 in FIG. 2 is a DNA molecular weight marker, lane 2 is 20% glycerol, methanol 10%, lane 3 is 20% glycerol, methanol 11%, lane 4 is glycerol 20% methanol 12%, lane 5 is glycerol 20% Methanol 13%, lane 6 is 20% glycerol, ethanol 13%, lane 7 is glycerol 20% ethanol 14%, lane 8 is glycerol 20% ethanol 15%, lane 9 is glycerol 20% ethanol 16%, lane 10 Is distilled water. The primers used in this example are 185s / 185as for A in FIG. 2, 5382s / 5382as for B in FIG. 2, 6174s / 6174as for C in FIG. 2, HPRTs / HPRTas in D for FIG. 2 and nucleotide sequences and PCR conditions Are shown in Table 1.

The human genomic DNA used in the PCR was 200 ng of the purified DNA from the peripheral blood by the proteinase K / phenol-chloroform method. The composition of the PCR reaction mixture was 2 μl of each concentration of glycerol + methanol solution glycerol + 2㎕ ethanol solution and 10mM Tris-HCl, pH 8.3, 40mM KCl, 1.5mM MgCl 2, 50mM / ㎖ BSA, each of 250 uM dNTP, sense primer, antisense primer 20 pmoles each were used.

Example  3: PCR Maximum amount of cryoprotectant solution

A sample (20 μl) was added to 20% glycerol + 12% methanol (hereinafter abbreviated as "AFA 1") and 20% glycerol + 15% ethanol (AFA 1) and AFA 2 (5 μl) did not affect PCR significantly (FIG. 3). 3 A was added to the lane 3, 2 μl of the lane 3 was added, 3 μl of the lane 4 was added, and 4 μl of the lane 5 was added. , Lane 6 is added to 5 μl, lane 7 is added to 6 μl, lane 8 is added to 7 μl, lane 9 is added to 8 μl, lane 10 is added to 9 μl, lane 11 is distilled water and lane 12 is negative control. The primers used for the PCR were A1s / H4as, and the base sequences and the PCR conditions are shown in Table 1.

Example  4: Cryoprotectant solution PCR On the sensitivity of detection

In order to investigate the effect of "AFA 1" and "AFA 2" on the detection sensitivity of PCR, diluted DNA at a certain concentration was compared with distilled water (control group). Lane 1 is DNA size marker, lane 2 is TBNA DNA 10 ng, lane 3 is TBNA DNA 1 ng, lane 4 is TB (lane 4) Lane 5 TB DNA 10 pg, lane 5 TB DNA 10 pg, lane 6 TB DNA 1 pg, lane 7 TB DNA 100 fg, lane 8 TB DNA 10 fg, lane 9 TB DNA 1 fg, lane 10 TB DNA 100 lag, lane 11 being negative control to be. ( TB What does DNA stand for? )

The primers used for PCR are TBs / TBas, and the nucleotide sequences and PCR conditions are shown in Table 1.

Example  5: stored in freezing agent Oligonucleotide  stability

A pair of primers were respectively dissolved in "AFA 1", "AFA 2" and distilled water (control) to examine the stability of oligonucleotides stored in "AFA 1" and "AFA 2" (30 days, 60 days, 90 days, and 120 days, respectively). PCR was performed to determine the reactivity of each of the three primers. Lanes 1, 2, 3, 4, and 5 in FIG. 5 indicate distilled water (control), lanes 6, 7, 8, 9 and 10 in "AFA 1" Lanes 1, 6 and 11 are 0 months, lanes 2, 7 and 12 are 1 month, lanes 3, 8 and 13 are 2 months, lanes 4, 9 and 14 are 3 months, lanes 5, 10 and 15 Was stored at -20 ° C for 4 months.

The primer used for PCR was 185s / 185as. The nucleotide sequence and the PCR conditions are shown in Table 1.

Example  6: Dielectric stored in freezing agent DNA Stability of

In order to investigate the stability of the genomic DNA stored in "AFA 1" at -20 ° C., the DNA was dissolved in distilled water and compared with the genomic DNA stored at 4 ° C. The result was dissolved in "AFA 1" In the case of stored genomic DNA, there was no problem in activity even after 6 months. Lanes 1, 2 and 3 in Fig. 6 were stored in distilled water, lanes 4, 5 and 6 were stored in distilled water and added with 2 l of "AFA 1" in PCR, and lanes 7, ".

The primers used for the PCR were A1s / H4as, and the base sequences and the PCR conditions are shown in Table 1.

Example  7: stored in an antifreeze PCR  Stability of the master mixture

To compare the stability of the PCR master mix (10X buffer, 10 mM dNTP, 5 U Taq DNA polymerase), the PCR master mix was prepared by dissolving in distilled water at 4 ° C and storing in AFA 1 and storing at -20 ° C for 3 weeks. The human genomic DNA was diluted to 1/10, 1/20, 1/50, and 1/100, respectively, and the detection sensitivity was examined. As a result, there was no problem in activity as shown in FIG. Lanes 1, 2, 3 and 4 were stored at -20 ° C after dissolving in "AFA 1", lanes 5, 6, 7 and 8 were dissolved in distilled water and stored at -35 ° C, lanes 9, 10, 11 and 12 were dissolved in distilled water and stored at 4 ° C for 1 week. Lanes 13, 14, 15 and 16 were dissolved in distilled water and stored at 4 ° C for 2 weeks. Lanes 17, 18, 19 and 20 were dissolved in distilled water Lanes 1, 5, 9, 13 and 17 were diluted 1/10 of human genomic DNA, lanes 2, 6, 10, 14 and 18 were diluted 1/20 of human genomic DNA Lanes 3, 7, 11, 15 and 19 are human genomic DNA diluted 1/50, lanes 4, 8, 12, 16 and 20 are human genomic DNA diluted 1/100.

The primers used for PCR are ACTs / ACTas, and the nucleotide sequences and PCR conditions are shown in Table 1.

Example  8: stored in freezing agent Reverse transcriptase  Stability of the master mixture

To compare the stability of the reverse transcriptase master mix (10X buffer, 10 mM dNTP, 400 U RTase, 100 U RNasin), the reactivity of the reverse transcriptase master mixture stored in 4 ° C and -20 ° C in distilled water and the reactivity of 20% glycerol and 12% (AFA 1) containing cryoprotectant 1 (AFA 1) containing 20% glycerol and 15% ethanol (hereinafter referred to as AFA 2) The activity decreased remarkably after one week. In the case of distilled water -20 ℃ and "AFA 1" -20 ℃ storage, there was no decrease in activity until 3 weeks. Lanes 1, 2, 3 and 4 were stored at -20 ° C after dissolving in distilled water, lanes 5, 6, 7 and 8 were stored at 4 ° C after dissolving in distilled water, lanes 9, 10, 11 and 12 were stored at -20 ° C after dissolving in "AFA 1", lanes 1, 5 and 9 were stored for 0 week, lanes 2, 6 and 10 for 1 week, lanes 3, 7 and 11 for 2 weeks, 4, 8 and 12 were stored for 3 weeks.

In order to compare the degree of decrease in activity, the reverse transcriptase master mixture stored at -20 ° C was dissolved in distilled water at 5, 10, After 20 and 30 times of freezing / thawing, the activity was examined and it was confirmed that the activity was completely lost from 1 time as shown in FIG. 9, lanes 1 and 2 were stored at -20 캜 after dissolving in "AFA 1 ", lanes 3 and 4 were frozen / thawed five times after dissolution in distilled water, lanes 5 and 6 were stored 10 times in freezing / 7 and 8 show 20 freeze / thaw cycles, and lanes 9 and 10 show 30 freeze / thaw cycles.

In the case of the reverse transcriptase master mixture, it can be seen that when stored at -20 ° C in "AFA 1", it can be used most stably without decreasing the activity.

For the RT / PCR reaction, HCV RNA purified from the patient's serum was used. The primers used for PCR were HCV RT / 1st s, HCV RT / 1st as, HCV 2nd s and HCV 2nd as, The PCR conditions are shown in Table 2.

Example  9: stored in freezing agent RNA Stability of

In order to compare the stability of RNA stored at -20 ° C in "AFA 1", the reactivity of the reverse transcriptase master mixture stored at -20 ° C in dissolution in distilled water was compared. As a result, as shown in FIG. 10A, At 1 "-20 ℃, there was no decrease in activity until 3 weeks. Lanes 1 and 2 of FIG. 10A are stored in "AFA 1", lanes 3 and 4 are stored in distilled water, and lane 5 is a negative control. However, as a result of comparison after 11 months, the activity was completely reduced when stored at -20 ° C. after dissolution in distilled water as shown in FIG. 10B. When stored at -20 ° C. after dissolving in "AFA 1" It was confirmed that there was no influence on the temperature. Lane 1 is the freshly extracted control RNA, lanes 2 and 3 are stored at -20 ° C in distilled water, lanes 4 and 5 are stored in "AFA 1", lanes 3 and 5 are lanes 2 and 4 RNA is diluted.

In order to compare the degree of decrease in activity, the RNA was stored at -20 ° C in 1, 5, 10, After 15 times of freezing / thawing, the activity was examined and it was confirmed that activity was completely lost from 1 time as shown in Fig. 11, lanes 1 and 2 are stored in "AFA 1", lanes 3 and 4 are stored in distilled water, followed by one freeze / thaw process, lanes 5 and 6 are freeze / thaw 5 times, lanes 7 and 8 are freeze / Thawing process 10 times, lanes 9 and 10 are repeated 15 times of freezing / thawing process.

For the RT / PCR reaction, purified HCV RNA was used from the patient's serum. Primers used for PCR were HCV RT / 1st s, HCV RT / 1st as, HCV 2nd s and HCV 2nd as, 2.

Example  10: Stability of the restriction enzyme stored in the freezing inhibitor

In order to compare the stability of the restriction enzymes stored in the cryoprotectant, five kinds of restriction enzymes such as AvaI, BamHI, EcoRI, HindIII and SalI were stored at -20 ° C in 50% glycerol, "AFA 1" and "AFA 2" 0 weeks, 2 weeks, and 6 weeks. The amount of each restriction enzyme used was 4 U for AvaI, 8 U for BamHI, 6 U for EcoRI and Hind III, and 8 U for Sal I, and the amount of each solution contained in 10 μl reaction solution was set to 1 μl. After incubation at 37 ° C for one hour, it was confirmed on 8% agarose gel. As a result of the 6-week study, it was confirmed that all the activities of 50% glycerol, "AFA 1" and "AFA 2" were not affected at all as in 12A, 12B and 12C. In Fig. 12, lanes 1, 6 and 11 are AvaI, lanes 2, 7 and 12 are BamHI, lanes 3, 8 and 13 are EcoRI, lanes 4, 9 and 14 are HindIII, lanes 5, 10 and 15 are SalI, 1, 2, 3, 4 and 5 are 50% glycerol, lanes 6, 7, 8, 9 and 10 are "AFA 1" and lanes 11, 12, 13, 14 and 15 are "AFA 2".

The DNA used for the restriction enzyme reaction was lambda DNA, and 150 ng was used in 10 μl reaction solution.

Example  11: Star activity of the restriction enzyme stored in the cryoprotectant

EcoR I was used to compare the star activity in 50% glycerol and "AFA 1", "AFA 2" solutions. 1 μl, 2 μl, and 3 μl of EcoR I contained in each solution were added to 10 μl reaction solution, reacted at 37 ° C for 30 minutes, and confirmed on 8% agarose gel. As a result of the experiment, 50% glycerol and "AFA 2" showed star activity from 2 μl and 3 μl showed "AFA 1" star activity. In case of "AFA 1" The star activity was significantly decreased compared to the control. 13, lane 1, 2 and 3 are 50% glycerol, lanes 4, 5 and 6 are "AFA 1", lanes 7, 8 and 9 are "AFA 2" 2, 5 and 8 were added in an amount of 2 μl, and lanes 3, 6 and 9 were added in 3 μl.

The DNA used for the restriction enzyme reaction was lambda DNA, and 100 ng was used in 10 μl reaction solution.

Example  12: When the cryoprotectant solution is in real time PCR ( Real - time PCR ) On the effect

To investigate whether the cryoprotectant solution used in the stability test in Examples 5 and 7 affects the reactivity and stability of real-time PCR, the probe labeled with oligonucleotide and fluorescent pigment and the PCR master mixture were added to "AFA 1" After the test was initiated and stored at -20 ° C for 6 months and 9 months, real-time PCR reactivity was confirmed.

Real-time PCR was performed by multiplex PCR on two targets. Two probes labeled with FAM and Cy5 were used in addition to two primers. The nucleotide sequence and PCR conditions are shown in Table 3.

The DNA used in the PCR is recombinant plasmid DNA containing a part of the nucleotide sequence of the HLA-B27 gene and the β-actin gene. The plasmid DNA isolated from the Escherichia coli culture broth using the separation and purification kit was quantified with a UV spectrophotometer The molecular weight was calculated and converted into the number of copies. For the real-time PCR reactivity measurement, a CFX96 Touch Real-Time PCR Detection System from Bio-Rad (USA) was used. The composition of the PCR reaction solution was 10X oligonucleotide mixture dissolved in "AFA 1" (5 μM HLA- And 2 μl of HLA-B27 as, HLA-B27 p at 2.5 uM concentration and IC p at IC su and IC as at 1 μM concentration, respectively, and 5 × PCR master mix (5 × PCR 5 μl dNTP mixture, 5 U Taq DNA polymerase). The template DNA was prepared by adding a positive control mixture (50,000copies / μl HLA-B27 and 5,000copies / μl of β-actin in solution in TE buffer Recombinant plasmid) solution and 12 占 퐇 of distilled water were added to adjust the final volume to 20 占 퐇.

Fig. 14A shows the results of the real-time PCR after starting the test, B at -20 ° C for 6 months, and C at -20 ° C for 9 months.

Claims (5)

A cryoprotectant composition capable of storing a biomaterial containing at least one of proteins or nucleic acids, which contains a combination of at least one of ethanol and methanol with glycerol, at a temperature below -40 占 폚 without freezing and without losing its activity.
The method according to claim 1,
Wherein the cryoprotectant composition comprises a cryoprotectant comprising a combination of at least one of 10-40% (v / v) ethanol or 10-30% (v / v) methanol and 10-40% (v / v) glycerol Composition.
The method according to claim 1,
Wherein the bio-material is at least one selected from the group consisting of polymerase, restriction enzyme, reverse transcriptase, DNA, RNA, oligonucleotide, and dNTP.
The method according to claim 1,
Wherein the cryoprotectant composition comprises a combination of at least one of 20-40% (v / v) ethanol or 20-30% (v / v) methanol and 20-40% (v / v) glycerol, Freezing agent composition which can be stably stored at a temperature not higher than 0 ° C and not higher than -40 ° C even when diluted to 1/2 hour.
(V / v) ethanol or 20-30% (v / v) methanol and 20-40% (v / v) glycerol, A cryoprotectant composition which can be stably stored at a temperature not higher than 0 ° C and not higher than -40 ° C even in the case of dilution, and at least one biomaterial of proteins and nucleic acids at a volume ratio of 1: 1.

Images