After running an agarose gel to isolate your PCR fragment, you will proceed with a clean-up procedure that is very similar to the direct PCR clean-up column, except the only thing you will be purifying is your fragment of interest. The alternative to a PCR clean-up column is band purification. There are some drawbacks to using PCR clean-up columns as they may not remove all your primers, template, or incorrect fragments. PCR clean-up columns will remove 100% of enzymes, salts, and remaining dNTPs.
The fastest and most common way to clean up PCR products is with PCR clean-up columns. Most cloning reactions perform better with purified DNA fragments. A little forethought now will likely save you time and money later. Others do less functionally, but essentially put your PCR product in the middle of a Multi-cloning site. Many are pre-configured for a specific purpose ranging from protein expression and purification to creating Gateway Entry clones. There are many choices of vectors when it comes to cloning PCR products. In addition, keeping the rounds of PCR to a minimum, below 30, lowers the risk of mutations. There are several PCR enzymes on the market with 20x or greater fidelity than traditional Taq polymerase which are recommended for use when performing PCR for cloning.
Mutations accumulate with each passing round of amplification so that by the end of 30 rounds of amplification, in which millions of base pairs are being copied per round, the odds of finding an unmutated product is less than 1 in 10. Choose the best PCR enzymeīasic Taq polymerase will introduce an error once every 10,000 base pairs replicated. In addition to good basic PCR habits, discussed in designing and performing PCR, there are some other specifics to bear in mind. TOPO cloning Cloning PCR Products Top Tips Depending on the ligase you use, you may need to incubate the ligation for several hours. However, the overhang is a single T/A at either end of the insert-vector junction and hence is not very stable. The insert can ligate into the vector in either orientation, which you can resolve when sequencing your candidate clones. Like restriction enzyme cloning, standard T/A cloning uses DNA Ligase to join the insert and vector. TA vectors are purchased as linear molecules with the 5’ T already added. Standard Taq polymerase adds this untemplated A, as do many others. TA cloning requires the use of PCR enzymes that add an untemplated A to the 3' end of a complete PCR product. While dedicated vectors are not unlimited, there are many TOPO and T/A vectors to choose from, and several allow you to progress very quickly to the next phase of your experiment. Both of these techniques allow you to clone your PCR product directly with minimal additional steps. TA and TOPO-TA cloning are two similar and highly effective ways to directly clone PCR products without using restriction enzymes. Restriction enzyme diagram for PCR cloning Cloning PCR Products without Restriction Enzymes
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