PCR product cloning is roughly divided into two categories, namely, flat-headed connections and sticky-head connections.
The flat head connection is the direct connection of the prepared flat head carrier to the flattened or flattened PCR product. The vector can be cut into a flat head with EcoR V or Sma I; after purification of the PCR product, it can be treated with DNA polymerase I for 30 min at 22 ° C (using the 3'→5' exonuclease activity of the enzyme and 5'→3' Polymerase activity). If the requirements are not high, the PCR product can also be left untreated. If Stratagene's pfu DNA polymerase or New England Biolabs' Vent DNA polymerase is used, the two enzymes have a 5'→3' proofreading capability, and the amplified PCR product is already flat and can be left unfinished. An obvious drawback of the flat-end connection is that the connection is inefficient, and even with the use of a very high unit of ligase, or the addition of PEG 8000 to the reaction system, the efficiency can only be increased to a very limited extent.
Sticky head connections can also be broadly classified into two categories. One type of glue head connection is a method that produces long, complementary, viscous ends on the support and PCR product. The most common method is to add a recognition sequence for a restriction enzyme at the 5' end of the primer. If the two primers use different restriction endonuclease recognition sequences, directional ligation can be achieved. The other class utilizes the characteristic of a protruding dAMP at the 3' end of the partial PCR product to construct a vector with a protruding dTMP at the 3' end. The method generally used is to digest the vector into a blunt end with a restriction enzyme, and treat it with Taq DNA polymerase for half an hour at 70 ° C or 72 ° C in a reaction system in which only one dNTP, dTTP is added (also It has been reported that treatment for 1 to 2 hours can improve the efficiency of cloning, so that the T reaction will be more thorough). The T-reaction can also be accomplished using a terminal transferase. Vector self-ligation and PCR product concatenation can be ignored. If you use ddTTP, the effect will be better. This method is generally called the addition of T/A method, which is 50 to 100 times more efficient than the flat connection.
I. TA Cloning of PCR Products 1. TA Cloning Construction Principle: TA Cloning System A commercial kit developed by Invitrogen (San Diego, CA) for the cloning and sequencing of PCR products. The principle is to use Taq enzyme to add a non-template-dependent A to the 3' end of the PCR product, while the T vector is a vector with a 3'T overhang, which can be quickly and step by step under the action of ligase. The PCR product was inserted directly into the multiple cloning site (MCS) of the plasmid vector.
2. Procedure (1) The ligation reaction is generally carried out in a sterilized 0.5 ml centrifuge tube.
(2) The 10 μl volume reaction system is as follows:
1 Take 1 μl of T vector (50 ng) and add equimolar PCR products.
2 Add 1 μl of 10×Buffer containing ATP, suitable unit of T4 DNA ligase, and make up to 10 μl with ddH2O.
(3) Centrifuge slightly, usually at 14-16 ° C water bath for 8-14 hr, or 4 ° C overnight.
(4) Transfection, blue and white spots are screened as before.
Second, matters needing attention
1. To obtain a TA clone of the gene of interest, the specificity of the PCR product is better.
2. The PCR product was purified by TA before cloning.
3. Prevent foreign DNA contamination during PCR product recovery and purification.
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