Team:TzuChiU Formosa/Discussion

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Revision as of 16:04, 19 October 2009

Discussion

digestion

　　The cause of unsuccessful digestion might be the few data after progress A-G PCR, or the concentration was not high enough.

TA cloning

　　Why using TA cloning? Because of its connecting way was “blunt end”, and that improves ligation’s efficiency. Furthermore, it is no need to do the digestion. But the main defect of this technique is that it doesn’t have concentrated ability, so in the next step it is still risky to disperse. Our experiment was also having other insert in it, which cause the misconnecting to the wrong site. In order to reduce the risk we have to raise the insert’s purity or take more single colonies to increasing the purity.

PSBlA3

　　When we started to transformations, we discovered that the colony still couldn’t be cultured. And we’ve figured out three reasons. First, at the heat shock time stage we using four times to test : 45(sec), 60(sec), 90(sec), 120(sec). And we found out that 60(sec) has better result than others, but compared to the paper’s result, it was still not going so well. So we speculated that it was also affected by the time when it was placed on the ice. Second, the time of the mixture of competent cell and plasmid on the ice might be too long (over 30 minutes). We have found in some protocol that only put it for five minutes. So we change it into five minutes, and the effect was very great. The last reason is the competent cell might be placed for a while which decreased the efficiency.

　　Due to the A-G sequence was supplied by France, the vector concentration and purity was unknown by us. We need to go through a series of purifying steps. After finish the A-G PCR, we discovered that it still included some unclear band. When we finish the whole purifying progress, it still had some unknown band. For the better purity and higher A-G concentration, we chose TA cloning and cloning PCR. Because of the previous experiment, TA cloning has been interfered by the unclear band. That cause TA cloning binding to the wrong insert. So we pick the other plate, and this time it was very lucky that we successfully got the correct result.

@@ Line 14: / Line 14: @@
-　　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.
+'''digestion'''
+　　The cause of unsuccessful digestion might be the few data after progress A-G PCR, or the concentration was not high enough.
-　　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.
+'''TA cloning'''
-　　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.
+　　Why using TA cloning? Because of its connecting way was “blunt end”, and that improves ligation’s efficiency. Furthermore, it is no need to do the digestion. But the main defect of this technique is that it doesn’t have concentrated ability, so in the next step it is still risky to disperse. Our experiment was also having other insert in it, which cause the misconnecting to the wrong site. In order to reduce the risk we have to raise the insert’s purity or take more single colonies to increasing the purity.
+'''PSBlA3'''
+　　When we started to transformations, we discovered that the colony still couldn’t be cultured. And we’ve figured out three reasons. First, at the heat shock time stage we using four times to test : 45(sec), 60(sec), 90(sec), 120(sec). And we found out that 60(sec) has better result than others, but compared to the paper’s result, it was still not going so well. So we speculated that it was also affected by the time when it was placed on the ice. Second, the time of the mixture of competent cell and plasmid on the ice might be too long (over 30 minutes). We have found in some protocol that  only put it for five minutes. So we change it into five minutes, and the effect was very great. The last reason is the competent cell might be placed for a while which decreased the efficiency.
+　　Due to the A-G sequence was supplied by France, the vector concentration and purity was unknown by us. We need to go through a series of purifying steps. After finish the A-G PCR, we discovered that it still included some unclear band. When we finish the whole purifying progress, it still had some unknown band. For the better purity and higher A-G concentration, we chose TA cloning and cloning PCR. Because of the previous experiment, TA cloning has been interfered by the unclear band. That cause TA cloning binding to the wrong insert. So we pick the other plate, and this time it was very lucky that we successfully got the correct result.