The TA Cloning System can be used for rapid, one-step insertion of PCR products directly into the multiple cloning site (MCS) of plasmid vectors.
Operation method
TA cloning of PCR products
Principle
PCR product cloning is broadly categorized into two types, namely, flat ligation and sticky ligation. Flathead ligation involves the direct ligation of a prepared flathead vector with a flattened or flattened PCR product. The vector can be cut into flats with EcoR V or Sma I. After purification, the PCR product can be run for 30 min at 22°C with DNA polymerase I (to take advantage of the 3'-to-5' exonuclease activity and the 5'-to-3' polymerase activity of the enzyme). The PCR products can be left untreated if the requirements are low. If Stratagene's pfu DNA polymerase or New England Biolabs' Vent DNA polymerase are used, which are 5'→3' proofreading enzymes, the amplified PCR product is already flat-ended and can be left untreated. An obvious disadvantage of flat-end ligation is that it is inefficient, and even with the use of very high units of ligase or the addition of PEG 8000 to the reaction system, only a limited increase in efficiency can be achieved. Sticky end ligations can also be broadly categorized into two groups. In one group, sticky end ligations are used to create long, complementary sticky ends on the vector and the PCR product by some method. The most common method is to add a restriction enzyme recognition sequence to the 5' end of the primer. If different restriction endonuclease recognition sequences are used for the two primers, targeted ligation can be achieved. Another approach is to construct vectors with a protruding dTMP at the 3' end of some PCR products, taking advantage of the fact that some PCR products have a protruding dAMP at the 3' end. This is generally accomplished by digesting the vector with a restriction endonuclease and treating it with Taq DNA polymerase at 70°C or 72°C for half an hour in a system that incorporates only one type of dNTP, i.e., dTTP (it has also been reported that treatment for one to two hours improves the cloning efficiency and makes the T-addition reaction more complete). Terminal transferase can also be used to complete the T-addition reaction. Vector self-association and PCR product crosstalk can be ignored. If ddTTP is used, the results are even better. This method, commonly referred to as plus-T/A cloning, is 50 to 100 times more efficient than flat-top ligation.
Materials and Instruments
PCR products Move 1、Linkage reaction is usually carried out in sterilized 0.5 ml centrifuge tubes. 2. The reaction system for a 10-μl volume is as follows: (1) Take 1 μl of T vector (50 ng) and add equal moles of PCR product. (2) Add 1 μl of 10× Buffer containing ATP and appropriate units of T4 DNA ligase and top up to 10 μl with ddH2O. (3) Slightly centrifuge, usually at 14-16°C in a water bath for 8-14hr, or 4°C overnight. (4) Transfection, blue and white spot screening as before. Caveat 1. To obtain a TA clone of the target gene, the PCR product must be of good specificity. 2. The PCR product is purified before TA cloning. 3. The PCR product is recovered and purified to prevent foreign DNA contamination. Common Problems An obvious drawback of flat-end ligation is the inefficiency of the ligation, which can only be improved to a very limited extent, even when very high units of ligase are used, or PEG 8000 is added to the reaction system. For more product details, please visit Aladdin Scientific website.
T4 DNA ligase ddH2O
Centrifuge Water bath