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A method of constructing T carriers

A carrier and enzyme cutting site technology, applied in the field of genetic engineering, can solve the problem of high background of non-recombinant transformants, and achieve the effect of improving quality and stability and excellent product characteristics

Inactive Publication Date: 2009-03-04
TSINGHUA UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] There is a potential problem whether XcmI or AhdI and its isoschizase are used to construct T vectors: partially digested pre-T vectors mixed in T vectors will lead to excessive background of non-recombinant transformants (Mead, D.A., Pey, N.K., Herrnstadt, C., Marcil, R.A., Smith, L.M., 1991. A universal method for the direct cloning of PCR amplified nucleic acid. Bio / Technology 9, 657-663; Harrison, J., Molly, P.L., Clark , S.J., 1994.Direct cloning ofpolymerase chain reaction products in an XcmI T-vector.Anal.Biochem.216,235-236)
However, a new problem brought about by the introduction of spacer DNA is that since circular plasmids move faster in agarose gel electrophoresis than linear plasmids of comparable molecular weight, undigested circular plasmids (pre-T vector, with spacer DNA) is often mixed with the T vector (without spacer DNA) whose molecular weight is smaller than itself, and finally causes the background of non-recombinant transformants to be too high

Method used

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Examples

Experimental program
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Effect test

Embodiment 1

[0041] Example 1, Construction of pSKΔAhd

[0042] Use the method of site-directed mutagenesis to silence the AhdI restriction site of the Amp resistance gene in the pBlueScript SK(-) vector to obtain pSKΔAhd. ) vector as a template, PCR amplification was carried out with Pfu DNA polymerase. Among them, the PCR amplification primers are (boxed C is the introduced mutation site (G in the original sequence), and the shaded part is the AhdI restriction site after mutation destruction):

[0043] AmpF: 5'-CTACGATACGGGAGGGCTTACCATCTGG-3';

[0044] AmpR: 5'-TTATCTACAC AGGCAAC-3';

[0045] The reaction system of PCR amplification is: 5U Pfu, 0.2mmol / L dNTPs, 1×Pfu buffer in 50μL reaction system, 10μmol / L of primers, about 5ng of template; the cycle program of PCR amplification is: 94℃ pre-denaturation for 5min , (94°C, 30S; 60°C, 30S; 72°C, 5min) × 30, 72°C for a final extension of 5min. After 1% agarose gel electrophoresis, the gel was cut to recover the PCR amplification produ...

Embodiment 2

[0049] Example 2. Introducing the AhdI box into the multiple cloning site of pSKΔAhd to construct the pre-T vector

[0050] Using the plasmid pENTR1A (purchased from Invitrogen) as a template, PCR amplification was performed with CcdB01F and CcdB01R primers. The reaction system of PCR amplification is: 5U Pfu, 0.2mmol / L dNTPs, 1×Pfu buffer in 50μL reaction system, 10μmol / L of primers, about 5ng of template; the cycle program of PCR amplification is: 94℃ pre-denaturation for 5min , (94°C, 30S; 60°C, 30S; 72°C, 1.5min) × 30, 72°C for a final extension of 5min. The PCR products were recovered by 1% agarose gel electrophoresis, and the recovered PCR product and pSKΔAhd plasmid were digested with SalI and XbaI respectively, and the digested products were recovered by 1% agarose gel electrophoresis. Establish the following ligation reaction system between the digested PCR product and the digested pSKΔAhd vector: 10 μL ligation system containing 3U ligase (Promega), 1× ligation buff...

Embodiment 3

[0068] Example 3, Preparation of pSKxx-T Series T Vectors and Analysis of TA Cloning Efficiency

[0069] 1. Preparation of T carrier

[0070] The pre-T vectors pSK01, 02, 03 and 04 were respectively digested with AhdI, and the enzyme digestion reaction system was as follows: 20 μL digestion system contained 3U AhdI endonuclease (purchased from NEB Company), 1×NEBuffer4, and about 1 μg plasmid. After enzyme digestion at 37°C for 3 hours, large fragments were recovered by 1% agarose gel electrophoresis. The large fragment recovered and purified by gel cutting is the corresponding T vector. The physical maps of pSK01-T, pSK02-T, pSK03-T and pSK04-T are as follows figure 2 , image 3 , Figure 4 with Figure 5 shown.

[0071] 2. Test TA cloning efficiency

[0072] Arabidopsis NCED3 (1.8kb, GenBank Accession No.AT3G14440) and DREB1A (680bp, GenBank Accession No.At4g25480) were amplified by PCR using Taq DNA polymerase with NCEDF and NCEDR as primers, respectively fragment. ...

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Abstract

A process for configuring the T carrier with higher Ta cloning efficiency includes preparing the T carrier precursor containing XcmI box or AhdI box, and severing the T carrier precursor by XcmI (or its isoschizomer) or AhdI (or its isoschizomer) to obtain T carrier.

Description

technical field [0001] The invention relates to a method for constructing a vector in the field of genetic engineering, in particular to a method for constructing a T vector. Background technique [0002] PCR (polymerase chain reaction) is a routine experimental technique in molecular biology. In most cases, it is necessary to clone the PCR amplification product into a plasmid vector for sequencing, in vitro transcription, in vitro translation and other operations on the PCR product. There are many methods for cloning PCR products, but the most direct and convenient method is TA cloning. The so-called TA cloning is to clone the PCR product with a single A tail at the 3' end to a T vector with a single T tail at the 3' end. The theoretical basis for TA cloning is that during PCR amplification, due to the template-independent terminal transferase activity of Taq polymerase, a single deoxynucleotide is added to the 3' end of the PCR product, and four deoxynucleosides are pref...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/63C12N15/64C12N15/66
Inventor 陈其军王学臣周海梦
Owner TSINGHUA UNIV
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