CN102628057B - Vector for non-background directed cloning of PCR products, preparation method thereof and application thereof - Google Patents
Vector for non-background directed cloning of PCR products, preparation method thereof and application thereof Download PDFInfo
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Abstract
The invention discloses a vector for non-background directed cloning of PCR products, a preparation method thereof and an application thereof. The vector is characterized in that an XcmI box is introduced before the vector screens and labels gene, and the XcmI box is characterized in that a protruded dT end is formed after XcmI enzyme digestion, an inactive ribosome binding site is formed before gene screening and labeling and after sequence splicing, and the screened and labeled gene cannot express during self-joining of the vector. The protruded dT end formed after the XcmI enzyme digestion of the vector prepared in the invention can be applied to TA cloning, and can be filled in by a T4DNA polymerase to form blunt ends, so as to be applied to blunt end joining; the method of the invention has the advantages of easy implementation, and low cost; and effects of no background and directed cloning are reached in the application of two cloning methods.
Description
Technical field
The invention belongs to gene engineering technology field, be specifically related to a kind of can be without the carrier of background directed cloning PCR product, also relate to a kind of can be without the preparation method of background directed cloning PCR product carrier, also relate to simultaneously a kind of can be without the application of background directed cloning PCR product carrier.
Background technology
PCR (polymerase chain reaction) is the experimental technique of the routine in molecular biology, and most gene Research on Functions etc. needs that all then the purpose fragment out is cloned into corresponding carrier by pcr amplification and studies.At present the method for clone PCR products has a variety ofly, comprises that traditional enzyme cuts interconnection technique, newly developed does not rely on the Gateway that enzyme is cut
System does not rely on the Topo cloning of T4 DNA ligase
And do not rely on cloning system (ligation-independent cloning, LIC) of ligase enzyme etc.However, build as cDNA library at some, the protein-interacting network detects, and transcriptional control network research etc. need in the extensive clone's of structure work, and above-mentioned these methods all have limitation separately.Enzyme is cut the method complex operation of connection, and the restriction of conditionality restriction endonuclease recognition site, Gateway
With Topo cloning
Running cost is higher, and the method for LIC depends on the complementary district of single stranded DNA of 10-15bp, thereby this zone easily forms secondary structure impact clone's efficient usually.Therefore, the simplest method still directly is connected the PCR product with corresponding carrier.
The direct connection carrier ordinary method of PCR product adopts TA clone scheme exactly, the terminal enzyme (DNA) activity of utilizing the Taq enzyme to have the template of not relying on adds single deoxynucleotide A at 3 ' end of PCR product, then the processing (comprise and add the dT tail and adopt specificity restriction endonuclease etc.) by certain method obtains outstanding dT on cloning vector, completes direct connection under the effect of T4DNA ligase enzyme by the TA complementation.Utilize this principle, developed now multiple business-like T carrier, by various transformations, the investigator has developed the T carrier of different selection markers at present, and the T carrier of high positive rate etc., but all with same dA tail, it is still a problem that exists at present that the purpose fragment is inserted positive and negative screening due to purpose fragment two ends.Although have at present by introducing the specificity base to the PCR product, synthesize different restriction endonuclease recognition sequences from the carrier sequence set after making it forward and oppositely being inserted into carrier, carry out the specificity restriction endonuclease to process can be largely upper insertion of removing fragment positive and negative by connecting product, but increased experimental procedure, complex operation particularly increases its workload in extensive clone's process.
The method of another PCR product Direct Cloning is that flat end connects.Because the Taq enzyme does not have 3 '-5 ' 5 prime excision enzyme activity, has relatively high mispairing rate in the PCR process, generally all adopt the high-fidelity DNA polymerase with 3 '-5 ' 5 prime excision enzyme activity to increase when transferring gene, the PCR product that obtains does not thus have outstanding A tail, only forms flat end products.The connection carrier of flat end products must be also flat end, and its carrier is that one in flat end connection procedure perplexs greatly from connecting.The processing such as carrier dephosphorylation can reduce carrier from the probability that connects, but the PCR primer need to pass through phosphatizing treatment thus, increased to a great extent experimental cost, problem due to carrier dephosphorylation efficient, clone's positive rate far can not reach the positive and negative screening of the flat end clone of 100%, PCR product insertion has also increased workload to extensive clone.
In sum, have the advantages such as simple to operate although the Direct Cloning of PCR product builds extensive clone, also need to be optimized to a great extent on colony screening.
Summary of the invention
The objective of the invention is to be to provide a kind of PCR product cloning carrier, this carrier can reach the purpose without background, directed cloning PCR product.
Another object of the present invention is the preparation method who has been to provide a kind of PCR product cloning carrier, and is easy to implement the method, with low cost, for supplying method is particularly cloned in gene clone on a large scale.
A further object of the present invention is the application that has been to provide a kind of PCR product cloning carrier, the TA that this carrier both can have been completed the PCR product connects the clone, can complete again flat end and connect the clone, and the application in two kinds of cloning process all can reach the effect without background, directed cloning.
In order to realize above-mentioned purpose, the present invention adopts following technical measures:
The cloning vector of the present invention's design is designed to resistant gene with PCR fragment insertion point, and the activated ribosome bind site of tool is not inner, can there be the mode of active ribosome bind site to screen correct clone with the carrier sequence set is synthetic by one section introducing of PCR fragment in clone's building process, reverse insertion all causes the ribosome bind site of selection markers gene not have activity with fragment and carrier is from connecting, thereby reaches the purpose without background, directed cloning.
Theoretical basis of the present invention is: the ribosome bind site that is positioned at gene start codon upstream 8 ± 3bp is the initial critical elements of expression of prokaryotic gene, and its sequence is comprised of the Nucleotide that is rich in GA.Ribosome bind site sequence before different genes is not quite similar, but its sequence GA content directly affects gene expression efficiency.When being mutated into TCCAGA as the ribosome bind site sequence by AGGAGA, gene is closed expression.according to above-mentioned theory, the activated ribosome bind site of not tool at resistant gene upstream from start codon 5bp place's introducing TCCAGA, and in the middle PCR fragment insertion point of introducing of TCC-AGA, PCR fragment 5 ' end is introduced GGA by primer, 3 ' end is introduced the AGG sequence by primer, PCR fragment forward will form the activated ribosome bind site of AGGAGA after being inserted into carrier thus, thereby start the expression of corresponding resistant gene, and the PCR fragment is oppositely inserted or carrier is not had a ribosome bind site of activity from all forming TCCAGA, be cloned in to contain on corresponding antibiotic flat board and all can not grow, get rid of thus the experiment reason can reach in theory the PCR product without the background directed cloning.
A kind of preparation method of the carrier without background directed cloning PCR product comprises the following steps:
1) comprise the preparation of intermediate carrier of the resistance screening gene of non-activity ribosome bind site: take universal support pUC18 (TAKARA company buy) as precursor, selection markers gene cat replacement lacZ α fragment wherein with comprising multiple clone site and non-activity ribosome bind site obtains intermediate carrier pBP1; Its concrete steps are:
A. utilize Auele Specific Primer to comprise Amp resistant gene, replicon, the carrier frame of lac promoter element take the pUC18 carrier as template with high-fidelity DNA polymerase KOD-Plus-Neo amplification; Amplification system is: 10 * PCR Buffer for KOD-Plus-Neo, 5 μ l, 2mM dNTPs 5 μ l, 25mM MgSO
43 μ l, 10 μ M forward primer 1 μ l, 10 μ M reverse primer 1 μ l, it is 50 μ l that pUC18 DNA 100ng, ultrapure water are added to last system, adds at last KOD-Plus-Neo 1U; Forward primer: SEQ ID No:2, reverse primer: SEQ ID No:3; Obtain the carrier frame fragment after utilizing PCR to reclaim the test kit purifying, then with the restriction endonuclease BglII of identification fragment two ends restriction site and KpnI, the carrier frame fragment is carried out enzyme and cut, and the recovery respective segments;
B. utilize Auele Specific Primer to use the reading frame of high-fidelity DNA polymerase KOD-Plus-Neo amplification cat gene take the pACYC184 plasmid as template; Amplification system is: 10 * PCR Buffer for KOD-Plus-Neo, 5 μ l, 2mM dNTPs 5 μ l, 25mM MgSO
43 μ l, 10 μ M forward primer 1 μ l, 10 μ M reverse primer 1 μ l, it is 50 μ l that pACYC184 DNA 100ng, ultrapure water are added to last system, adds at last KOD-Plus-Neo 1U, forward primer: SEQ ID No:4, reverse primer: SEQ ID No:5; Utilize PCR to reclaim the test kit purifying, utilize the restriction endonuclease BglII at reading frame two ends of identification cat gene and KpnI to carry out enzyme to fragment and cut, and utilize DNA gel to reclaim test kit and reclaim fragment after enzyme is cut;
C. the fragment after the recovery of step a and step b acquisition is mixed with mol ratio at 1: 3, form closed hoop under the effect of T4 DNA ligase, after change the bacillus coli DH 5 alpha competent cell over to, obtain positive colony with Amp screening, therefrom extract plasmid and order-checking evaluation, obtain intermediate carrier pBP1;
2) a kind of preparation method of the carrier without background directed cloning PCR product:
A. design, synthetic XcmI box: synthetic XcmI box, its feature comprises 5 ' end restriction endonuclease recognition site, 5 ' end XcmI recognition site, the lac promotor, ccdB bears the selection markers gene, 3 ' end XcmI recognition site, 3 ' end restriction endonuclease recognition site etc., last base of point of contact in 5 ' end XcmI site is T, 3 of T fronts base does not contain A and G, last base of point of contact in 3 ' end XcmI site is A, 3 bases of A front do not contain A and G, two of A back base discord T or C, and the point of contact A in 3 ' end XcmI site and the distance between selection markers gene start codon ATG are between 7-9bp, the synthetic XcmI box sequence of design is seen sequence list SEQ ID No:1,
B. the XcmI box is carried out enzyme with PstI+SphI and cut, and utilize DNA gel to reclaim test kit and reclaim fragment after enzyme is cut;
C. intermediate carrier pBP1 is cut with the PstI+SphI enzyme, and utilize DNA gel to reclaim test kit and reclaim fragment after enzyme is cut;
D. the intermediate carrier pBP1 after the XcmI box that enzyme is cut back to close and enzyme cut back to close mixes take suitable proportion (mol ratio was as 3: 1), form the closed hoop analysis under the effect of T4DNA ligase enzyme, after change intestinal bacteria DB3.1 competent cell over to, obtain positive colony with the Amp screening, therefrom extract plasmid and order-checking evaluation, obtain purpose carrier pDNB1.Contain the XcmI box of synthetic before the selection markers gene reading frame of the carrier that obtains, the nucleotides sequence of XcmI box is classified as shown in SEQ ID NO.1.
Step 1 wherein) the described carrier pUC18 that sets out can select similar various cloning vector (as serial in pUC19, pUC118, pUC119, pBlueScript II SK, pGEM series, pMD etc.), and the selection markers gene of wherein selecting can be with other selection markers genes (as neo, tetR etc.) that do not comprise on the carrier that sets out.
Step 2) enzyme described in A is cut the XcmI box and can be realized that enzyme cuts the box that produces outstanding T end or flat end and replace with other, and its sequence signature satisfies enzyme and cuts after the connecting distance between resistant gene initiator codon ATG is assembled into non-activity before 5-7bp ribosome bind site.
The purpose carrier pDNB1 of the present invention's design need to breed in intestinal bacteria DB3.1, connects product in clone's building process and transforms common intestinal bacteria.
The application of a kind of carrier in PCR product TA connection clone the steps include:
1) cut purpose carrier pDNB1 with the XcmI enzyme, reclaim the T carrier that large carrier segments namely obtains directly to carry out the TA clone;
2) the specific forward and reverse primer of design amplified fragments is introduced GGA with forward primer 5 ' end, and 5 ' end of reverse primer is introduced CCT;
3) adopt Taq enzyme PCR reaction to amplify the PCR fragment of specific two ends band A tail;
4) with the PCR fragment and step 1 of two ends band A tail) in the T carrier that obtains be connected, transform bacillus coli DH 5 alpha, adopt two kinds of resistant genes on carrier to screen;
5) the picking clone carries out the PCR checking, detects the positive and negative and positive rate of Insert Fragment.
The application of a kind of carrier in the flat end of PCR product connects the clone the steps include:
1) cut purpose carrier pDNB1 with the XcmI enzyme, reclaim large carrier segments and namely obtain the T carrier, process this T carrier with the T4DNA polysaccharase, fill the dT tail, form flat terminal fragment, namely obtain directly to carry out the carrier that the flat end of PCR product connects.
2) the specific forward and reverse primer of design amplified fragments is introduced GGA with forward primer 5 ' end, and 5 ' end of reverse primer is introduced CCT;
3) adopt high-fidelity enzyme PCR reaction to amplify specific flat end PCR fragment;
4) with flat end PCR fragment and step 1) carrier that obtains is connected, and transforms bacillus coli DH 5 alpha, adopts two kinds of resistant genes on carrier to screen;
5) the picking clone carries out the PCR checking, detects the positive and negative and positive rate of Insert Fragment.
Step 1) the dT tail is removed and can be selected other modifying enzymes that can reach said function to replace.The present invention compared with prior art has the following advantages and effect:
1. the carrier of the present invention's preparation forms outstanding dT end and can be applied to the TA clone after the XcmI enzyme is cut, the dT end also can fill by the T4DNA polysaccharase and form flat end, be applied to flat end and connect, the application in two kinds of cloning process all can reach the effect without background, directed cloning.
2. the carrier of the present invention's design can prepare amplification voluntarily, easy to implement the method, with low cost, complete the quick clone of PCR product with the carrier of the present invention's design, positive rate reaches 100%, and can distinguish the forward and reverse of PCR fragment insertion, has greatly simplified the step of clone operations and screening, greatly reduce experimental cost, advantage is especially outstanding in high-flux clone builds.
Description of drawings
Fig. 1 is a kind of intermediate carrier pBP1 carrier collection of illustrative plates schematic diagram.
Fig. 2 is a kind of purpose carrier pDNB1 carrier collection of illustrative plates schematic diagram.The grey box representative is cut out next XcmI box; White box represents the cat reading frame.
Embodiment
Example 1:
Intermediate carrier pBP1 builds:
1) utilize Auele Specific Primer (forward primer: SEQ ID No:2, reverse primer: SEQ ID No:3) increasing comprises Amp resistant gene, replicon with high-fidelity DNA polymerase KOD-Plus-Neo (TOYOBO company purchase) for template take pUC18 carrier (TAKARA company buy), (amplification system is the carrier frame of lac promoter element: 10 * PCR Buffer for KOD-Plus-Neo, 5 μ l, 2mM dNTPs 5 μ l, 25mM MgSO
43 μ l, 10 μ M forward primer 1 μ l, 10 μ M reverse primer 1 μ l, pUC18 DNA 100ng, it is 50 μ l that ultrapure water is added to last system, add at last KOD-Plus-Neo 1U), obtain the carrier frame fragment after utilizing business-like PCR to reclaim test kit (Beijing hundred Tyke Bioisystech Co., Ltd) purifying, then with the restriction endonuclease BglII of identification fragment two ends restriction site (TAKARA company buy) and KpnI (TAKARA company buy), the carrier frame fragment is carried out enzyme and cut, and the recovery respective segments;
2) utilize Auele Specific Primer (forward primer: SEQ ID No:4, reverse primer: SEQ ID No:5) (amplification system is as 10 * PCR Buffer for KOD-Plus-Neo, 5 μ l with the reading frame of high-fidelity DNA polymerase KOD-Plus-Neo (TOYOBO company purchase) the cat gene that increases for template take pACYC184 plasmid (New England Biolabs company buy), 2mM dNTPs 5 μ l, 25mM MgSO
43 μ l, 10 μ M forward primer 1 μ l, 10 μ M reverse primer 1 μ l, pACYC184 DNA 100ng, it is 50 μ l that ultrapure water is added to last system, add at last KOD-Plus-Neo 1U), utilize business-like PCR to reclaim test kit (Beijing hundred Tyke Bioisystech Co., Ltd) purifying, utilizing the restriction endonuclease BglII (TAKARA company buy) at the reading frame two ends of identification cat gene and KpnI (TAKARA company purchase) to carry out enzyme to fragment cuts, and utilize the commercialization DNA gel to reclaim test kit (Beijing hundred Tyke Bioisystech Co., Ltd) and reclaim fragment after enzyme is cut,
3) with step 1) and step 2) fragment after the recovery that obtains mixes take suitable proportion (mol ratio was as 1: 3), form the closed hoop analysis under the effect of T4 DNA ligase, after change bacillus coli DH 5 alpha (TAKARA company buy) competent cell over to, obtain positive colony with the Amp screening, therefrom extract plasmid and order-checking evaluation, obtain intermediate carrier pBP1 (Fig. 1).
Example 2:
The structure of a kind of carrier pDNB1 without background directed cloning PCR product:
XcmI box about 1) design, synthetic 800bp, it comprises PstI recognition site, XcmI recognition site, lac promotor, and ccdB bears the features such as selection markers gene, XcmI recognition site, SphI recognition site, and sequence is seen sequence list SEQ ID No:1;
2) the XcmI box is carried out enzyme with PstI (TAKARA company buy) and SphI (TAKARA company buy) and cut, and utilize the commercialization DNA gel to reclaim fragment after test kit (Beijing hundred Tyke Bioisystech Co., Ltd) recovery enzyme is cut;
3) intermediate carrier pBP1 is cut with PstI (TAKARA company buy) and SphI (TAKARA company buy) enzyme, and utilize the commercialization DNA gel to reclaim fragment after test kit (Beijing hundred Tyke Bioisystech Co., Ltd) recovery enzyme is cut;
4) the intermediate carrier pBP1 after the XcmI box that enzyme is cut back to close and enzyme cut back to close mixes take suitable proportion (mol ratio was as 3: 1), form the closed hoop analysis under the effect of T4DNA ligase enzyme, after change intestinal bacteria DB3.1 (Invitrogen company buy) competent cell over to, obtain positive colony with the Amp screening, therefrom extract plasmid and order-checking evaluation, obtain purpose carrier pDNB1 (Fig. 2).
Example 3:
A kind of application of carrier on TA connection clone without background directed cloning PCR product:
1) process the pDNB1 plasmid with restriction enzyme XcmI (New England Biolabs company buy), the 3h rear electrophoresis reclaims the linear plasmid carrier that obtains the 2.5kb left and right, be called after pDNB-T, be the T carrier that can directly carry out PCR fragment TA clone;
2) utilize Auele Specific Primer (forward primer: SEQ ID No:6, reverse primer: SEQ ID No:7) take e. coli k12 (professor Qi Qingsheng of Shandong University is so kind as to give) strain gene group as template, (amplification system is: 10 * Taq Buffer, 5 μ l to amplify the SD-lacZa-1 fragment of 280bp left and right with Taq enzyme (buying from Beijing ancient cooking vessel state biotechnology limited liability company), 2mM dNTPs 5 μ l, 10 μ M forward primer 1 μ l, 10 μ M reverse primer 1 μ l, e. coli k12 genome 100ng, it is 50 μ l that ultrapure water is added to last system, add at last Taq enzyme 1U), obtain the carrier frame fragment after utilizing business-like PCR to reclaim the test kit purifying,
The SD-lacZa-1 that 3) will reclaim purifying mixes (mol ratio was as 5: 1) with pDNB-T take suitable proportion, use the T4 DNA ligase, connect product and transform bacillus coli DH 5 alpha (TAKARA company buy), coating LB+Amp+Cm+Xgal+IPTG is dull and stereotyped, 37 ℃ of overnight incubation;
4) transformed clone grows 102 clones on the flat board that contains IPTG and Xgal, all clones all present locus coeruleus, prove that the equal forward of all fragments inserts, and positive rate reaches 100%, 20 clones of picking carry out the PCR the result and show that all the clone inserts for the SD-lacZa-1 forward, and clone's positive rate reaches 100%.
Example 4:
A kind of application of carrier on flat end connection clone without background directed cloning PCR product:
1) prepare the pDNB-T carrier according to the method in example 3;
2) containing with the T4DNA polysaccharase dT tail that fills pDNB-T under the condition of dNTP, forming pDNB-B;
3) utilize Auele Specific Primer (forward primer: SEQ ID No:6, reverse primer: SEQ ID No:7) take the e. coli k12 strain genome as template, (amplification system is: 10 * PCR Buffer for KOD-Plus-Neo, 5 μ l to amplify the SD+lacZa-2 fragment of 280bp left and right with high-fidelity DNA polymerase KOD-Plus-Neo (TOYOBO company buy), 2mM dNTPs 5 μ l, 25mM MgSO
43 μ l, 10 μ M forward primer 1 μ l, 10 μ M reverse primer 1 μ l, it is 50 μ l that e. coli k12 genome 100ng, ultrapure water are added to last system, adds at last KOD-Plus-Neo 1U), obtain the carrier frame fragment after utilizing PCR to reclaim the test kit purifying;
The SD+lacZa-2 that 4) will reclaim purifying mixes (mol ratio was as 5: 1) with pDNB-B take suitable proportion, use the T4DNA ligase enzyme, connect product and transform bacillus coli DH 5 alpha (TAKARA company buy), coating LB+Amp+Cm+Xgal+IPTG is dull and stereotyped, 37 ℃ of overnight incubation;
5) transformed clone grows 480 clones on the flat board that contains IPTG and Xgal, all clones all present locus coeruleus, prove that the equal forward of all fragments inserts, and positive rate reaches 100%, 20 clones of picking carry out the PCR the result and show that all the clone inserts for the SD-lacZa-2 forward, and clone's positive rate reaches 100%.
SEQUENCE LISTING
＜110〉Wuhan Virology Institute,Chinan academy of Sciences
＜120〉a kind of can be without carrier and preparation and the application of background directed cloning PCR product
＜130〉a kind of can be without carrier and preparation and the application of background directed cloning PCR product
<160> 7
<170> PatentIn version 3.1
<210> 1
<211> 813
<212> DNA
＜213〉synthetic
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aaactgcagc cattcctaag ctgggtctca tgagcggata catatttgaa tgtatttaga 60
aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct gacgtctaag 120
aaaccattat tatcatgaca ttaacctata aaaataggcg tatcacgagg ccctttcgtc 180
tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 240
cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 300
ttggcgggtg tcggggctgg cttaaggtac cttatattcc ccagaacatc aggttaatgg 360
cgtttttgat gtcattttcg cggtggctga gatcagccac ttcttccccg ataacggaga 420
ccggcacact ggccatatcg gtggtcatca tgcgccagct ttcatccccg atatgcacca 480
ccgggtaaag ttcacgggag actttatctg acagcagacg tgcactggcc agggggatca 540
ccatccgtcg cccgggcgtg tcaataatat cactctgtac atccacaaac agacgataac 600
ggctctctct tttataggtg taaaccttaa actgcatatg tatatctcct tcttaaagtt 660
aaacaaaatt atttgaattc tccacacaac atacgagccg gaagcataaa gtgtaaagcc 720
tggggtgcct aatgagtgag ctaactcaca ttaattgcgt tgcgctcact gcccgctttc 780
cagtcgggaa ccattccaga tctggcatgc ttt 813
<210> 2
<211> 79
<212> DNA
＜213〉synthetic
<400> 2
tttagatcta agcttgcgca tgcacagctg cagtcgacgg atccatatga attctccaca 60
caacatacga gccggaagc 79
<210> 3
<211> 40
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＜213〉synthetic
<400> 3
aaaggtacct taagccagcc ccgacacccg ccaacacccg 40
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gcgagatctg agaaaaaaat cactgg 26
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attggtacct tacgccccgc cctgc 25
<210> 6
<211> 38
<212> DNA
＜213〉synthetic
<400> 6
ggagaaggaga tatacatatg accatgatta cggattc 38
<210> 7
<211> 23
<212> DNA
＜213〉synthetic
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ccttcagacg acagtatcgg cct 23
Claims (3)
1. the preparation method of a recombinant vectors pDNB1, the steps include:
A. prepare intermediate carrier pBP1:
A. utilize Auele Specific Primer to comprise Amp resistant gene, replicon, the carrier frame of lac promoter element take the pUC18 carrier as template with high-fidelity DNA polymerase KOD-Plus-Neo amplification; Amplification system is: 10 * PCR Buffer for KOD-Plus-Neo5 μ l, 2mM dNTPs5 μ l, 25mM MgSO
43 μ l, 10 μ M forward primer 1 μ l, 10 μ M reverse primer 1 μ l, it is 50 μ l that pUC18DNA100ng, ultrapure water are added to last system, adds at last KOD-Plus-Neo1U; Forward primer: SEQ ID No:2, reverse primer: SEQ ID No:3; Obtain the carrier frame fragment after utilizing PCR to reclaim the test kit purifying, then with the restriction endonuclease BglII of identification fragment two ends restriction site and KpnI, the carrier frame fragment is carried out enzyme and cut, and recovery carrier frame fragment;
B. utilize Auele Specific Primer to use the reading frame of high-fidelity DNA polymerase KOD-Plus-Neo amplification cat gene take the pACYC184 plasmid as template; Amplification system is: 10 * PCR Buffer for KOD-Plus-Neo5 μ l, 2mM dNTPs5 μ l, 25mM MgSO
43 μ l, 10 μ M forward primer 1 μ l, 10 μ M reverse primer 1 μ l, it is 50 μ l that pACYC184DNA100ng, ultrapure water are added to last system, adds at last KOD-Plus-Neo1U, forward primer: SEQ ID No:4, reverse primer: SEQ ID No:5; Utilize PCR to reclaim the test kit purifying, utilize the restriction endonuclease BglII at reading frame two ends of identification cat gene and KpnI to carry out enzyme to fragment and cut, and utilize DNA gel to reclaim test kit and reclaim fragment after enzyme is cut;
C. the fragment after the recovery of step a and step b acquisition is mixed with mol ratio 1:3, form closed hoop under the effect of T4DNA ligase enzyme, after change the bacillus coli DH 5 alpha competent cell over to, obtain positive colony with Amp screening, therefrom extract plasmid and order-checking evaluation, obtain intermediate carrier pBP1;
B. build purpose carrier pDNB1:
A. design, synthesize XcmI box nucleotide sequence, the nucleotides sequence of XcmI box is classified as shown in SEQ ID NO.1;
B. the XcmI box is carried out enzyme with PstI and SphI and cut, and utilize DNA gel to reclaim test kit and reclaim fragment after enzyme is cut;
C. intermediate carrier pBP1 is cut with PstI and SphI enzyme, and utilize DNA gel to reclaim test kit and reclaim fragment after enzyme is cut;
D. the intermediate carrier pBP1 after the XcmI box that enzyme is cut back to close and enzyme cut back to close mixes with mol ratio 3:1, form closed hoop under the effect of T4DNA ligase enzyme, after change intestinal bacteria DB3.1 competent cell over to, obtain positive colony with the Amp screening, therefrom extract plasmid and order-checking evaluation, obtain carrier.
2. the application of the prepared carrier of the preparation method of recombinant vectors pDNB1 claimed in claim 1 in PCR product TA clone.
3. the application of the prepared carrier of the preparation method of recombinant vectors pDNB1 claimed in claim 1 in the flat end of PCR product connects the clone.
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| CN103757039B (en) * | 2013-03-27 | 2016-04-27 | 广州赛哲生物科技股份有限公司 | A kind of multi-functional cloning vector and using method thereof |
| CN104313044B (en) * | 2014-09-30 | 2017-04-26 | 天根生化科技（北京）有限公司 | Zero-background cloning vector as well as preparation method and application thereof |
| CN110540998B (en) * | 2018-05-28 | 2024-01-05 | 深圳华大生命科学研究院 | Method and reagent for quickly constructing multivalent antibody expression vector |
| CN114134091B (en) * | 2021-11-08 | 2024-04-12 | 中国科学院武汉病毒研究所 | Identification method and application of bacterial genome insulation sites |
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|---|---|---|---|---|
| CN101503698A (en) * | 2009-03-06 | 2009-08-12 | 西南大学 | Non-false positive T vector and preparation |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101503698A (en) * | 2009-03-06 | 2009-08-12 | 西南大学 | Non-false positive T vector and preparation |
Non-Patent Citations (4)
| Title |
|---|
| DNA cloning using in vitro site-specific recombination.;Hartley JL et al;《Genome Res》;20001231(第10期);全文 * |
| Hartley JL et al.DNA cloning using in vitro site-specific recombination..《Genome Res》.2000,(第10期),全文. |
| 一种提高PCR产物定向克隆效率的新方法;余传信等;《生物技术》;19971231;第5卷(第7期);全文 * |
| 余传信等.一种提高PCR产物定向克隆效率的新方法.《生物技术》.1997,第5卷(第7期),全文. |
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