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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