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Team:TzuChiU Formosa/Discussion
From 2009.igem.org
Discusion
The reason cause of unsuccessful digestion may be the data after progress A-G PCR, the concentration was not high enough, or the digestion didn’t take sufficient quantity, and cause the digestion failed.
Why we use TA cloning? Because of TA cloning connection’s way was blunt end, and that cause ligation efficiency going well. Furthermore, we don’t have to do the digestion. But the mainly defect of this technique is that don’t have concentrated ability, so in the next step it still have risk to disperse. Our experiments including other insert in it, and to cause it connect to the wrong site. In order to reduce the risk we have to raise the insert purity up or pick more single colony to increasing the purity.
When we start to transformations, we discovery that colony still couldn’t culture. And we discuss it may have three reasons. First, at the heat shock time stage we using four time to test it: 45(sec), 60(sec), 90(sec), 120(sec). And we found out that 60(sec) have better result than others, but compared to the paper’s result it still not going so well. So we speculated it was related with the time placed at the ice. Second, the time we put the mixture of competent cell and plasmid at the ice may to long (over 30 minutes) then we found in some protocol only put it five minutes. And we testing, the effect was very great, and successfully. The last reason was that the competent cell may place for a while so the efficiency may decrease.
Owing to the A-G sequence was supply by France institute, the vector concentration and purity was unknown. We need to finish a series of purity step. After finish the A-G PCR, we discover that it still includes some unclear band. When we finish the whole progress it still had some unknown band. For the successful purity and increasing A-G concentration, we chose TA cloning and cloning PCR. Because of the front experiment, TA cloning has been interfered with unclear band. That cause TA cloning had bind to wrong insert. So we pick others plates, and this time we successfully got the correct result.
