我的载体构建心得（My carrier construction experience）.docVIP

我的载体构建心得（My carrier construction experience） My carrier construction experience ---------aqi96 It took me 3 months to build a carrier. It was torture. Luckily, I had a good attitude. If you want to ask, why? It took so long to build, and indeed, for a person skilled in molecular biology, it should be 2-3 weeks Almost. It took so long, first of all, because I was totally new to the experiment. Ive been in the clinic for several years now Read the professional masters degree in molecular biology, reading in school is hazy, relatively poor foundation. Secondly, doing experiments There were no special people who came to me, so the process of doing it was full of hardships. Now the construction is finally successful, Finally relieved, but behind the road is still long, it is said to be more difficult, go step by step. The process of building my carrier and Write out the problems, hoping to give some comrades such as me some inspiration, I wish you success as soon as possible test. First, lessons learned in the PCR process In molecular cloning and construction of expression vectors, the target fragment must end with the sequence in the genebank All in all, so in the PCR process, enzymes should be selected first by high fidelity enzymes, especially when targeting This is especially true for periods of longer periods. This reduces mismatches in the PCR process. High fidelity enzymes are often correct The annealing temperature is required. For the general enzyme, the annealing temperature is Tm-5 degrees, but a high fidelity enzyme such as NEB The annealing temperature is Tm, +3 degrees. PCR cycle number should not be too large, 20-30 can. I used to use regular When the enzyme was amplified by PCR, the non specific bands appeared, and the T-A clones were sequenced and found to be mismatched, And its always different. Two, PCR products were not successful after direct enzyme digestion When you get the PCR product, you first purify, cut and purify the enzyme and then recycle i