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Method for enhancing DNA (deoxyribonucleic acid) ligase ligation efficiency by using polyethyleneimine

A technology of DNA ligase and polyethyleneimine, which is applied in the field of genetic engineering to achieve the effects of increasing the connection probability, increasing the relative concentration and solving the low connection efficiency

Active Publication Date: 2016-04-20
HUAQIAO UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

So far, there is no report on the use of electropositive polymers to effectively improve the ligation efficiency of T4DNA ligase at home and abroad.

Method used

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  • Method for enhancing DNA (deoxyribonucleic acid) ligase ligation efficiency by using polyethyleneimine
  • Method for enhancing DNA (deoxyribonucleic acid) ligase ligation efficiency by using polyethyleneimine
  • Method for enhancing DNA (deoxyribonucleic acid) ligase ligation efficiency by using polyethyleneimine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1 DNA target fragment preparation (fragment A)

[0037] 1. The PCR system (100ul system) is as follows

[0038] PUC19-fragment A (6ng / ul)

2ul

Primer 1 - Fragment A Forward Primer

1ul

Primer 2 - fragment A reverse primer

1ul

2×Master Mix

50ul

ddH2O

46ul

[0039] Primer 1 sequence: gtgaattcgagctcggtacccggaattcTTAGTTTACCGCGTCTT.

[0040] Primer 2 sequence: TgcaggtcgactctagaggatccatatgAATAAATCTCAATTGATCG.

[0041] Among them: gaattc is the EcoRI restriction site, Catatg is the NdeI restriction site.

[0042] 2. Carry out PCR reaction according to the following conditions: 95°C for 1min; 34 cycles of 95°C for 30s, 57°C for 30s, and 72°C for 30s; 72°C for 5min; 4°C∞.

[0043] 3. Use the Gel Recovery Kit to recover and purify Fragment A. Gel-recovered product detection: take 3ul of gel-recovered product, use 1% agarose gel as the electrophoresis support medium, electrophoresis at 1×TAE and 90V voltage ...

Embodiment 2

[0046] Embodiment two double digestion fragment A and PET22b plasmid

[0047] 1NdeI digestion

[0048] 1.1 NdeI restriction fragment A system

[0049]

[0050]

[0051] NdeI digestion PET22b plasmid system

[0052] wxya 2 o

21ul

Fast Digest Buffer

4ul

PET22b

33ul

Fast Digest Enzyme (NdeI)

2ul

60ul

[0053] 1.2 Carry out enzyme digestion reaction according to the following procedure: 37°C for 1h30min, 65°C for 5min, 4°C∞.

[0054] 1.3 Use the rapid purification and recovery kit to recover the digested product once.

[0055] 2EcoRI digestion

[0056] 2.1 EcoRI Fragment A System

[0057] wxya 2 o

29ul

Fast Digest Buffer

4ul

Fragment A

25ul

Fast Digest Enzyme (EcoRI)

2ul

60ul

[0058] EcoRI Digestion PET22b Plasmid System

[0059] wxya 2 o

21ul

Fast Digest Buffer

4ul

PET22b

33ul

Fast Digest ...

Embodiment 3

[0064] In Example 3, different concentrations of polyethyleneimine were added to the connection system and converted

[0065] 1T4Sticky-endLigation

[0066] 1.1 Add different concentrations of PEI 0.5ul to the T4 ligase system (20ul).

[0067]

[0068] 1.2 The connection procedure is as follows: 1h at 22°C, 10min at 65°C, ∞ at 4°C.

[0069] 2 Transformation: Take 10ul of the connection solution and add 100ul of BL21 (DE3) competent cells, ice bath for 30min, 42°C water bath for 75s, ice bath for 5min, then place in a clean bench and add 300ul of SOC medium, shake the bacteria at 37°C and 225rpm for 1h, Take 150 ul of the bacteria solution and apply it to the amp plate, and cultivate overnight at 37°C.

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Abstract

The invention provides a method for enhancing DNA (deoxyribonucleic acid) ligase ligation efficiency by using polyethyleneimine, which comprises the following steps: designing primers with enzyme digestion sites for the target DNA segment; carrying out PCR (polymerase chain reaction) to obtain the target DNA segment with the enzyme digestion sites on both ends; carrying out enzyme digestion to obtain a linearized vector and target DNA segment to be ligated; preparing a polyethyleneimine solution for later use; adding a preset volume of polyethyleneimine solution into the ligation system to carry out ligation reaction; and carrying out transformation and cloning in Escherichia coli to obtain the clone of the recombinant DNA molecule. The method can obviously enhance the ligation efficiency of the T4 DNA ligase, and fills up the blank in the field of application of electropositive high-molecular polymers (polyethyleneimine) in enhancing T4 DNA ligase ligation efficiency.

Description

【Technical field】 [0001] The invention belongs to the technical field of genetic engineering, and relates to a method for improving the connection efficiency of T4 DNA ligase by using an electropositive polymer polyethyleneimine (PEI). 【Background technique】 [0002] In vitro DNA recombination technology refers to the process of linking two or more DNA fragments from the same or different sources into a new recombinant DNA molecule in vitro, and transforming it into a recipient bacteria for autonomous replication, and finally screening and identifying the correct clone. This technique is the basic technique in genetic engineering. [0003] The basic procedures of in vitro DNA recombination technology include: acquisition of exogenous target DNA fragments, vector selection, connection, transformation and cloning. The linking step is a key step in obtaining recombinant DNA. The most commonly used method of connection is under the action of DNA ligase, with Mg 2+ In the liga...

Claims

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Application Information

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IPC IPC(8): C12N15/70C12N15/66
CPCC12N15/66C12N15/70C12N2800/101
Inventor 戚智青刘楠辛侯丹
Owner HUAQIAO UNIVERSITY