Aequorin-GFP coding Sequence containing plasmid was kindly provide by French scientist() We use PCR to Pst1 site at both ends of Aequorin-GFP coding Sequence. Then we insert Aequorin-GFP lst into TA vector to have easier cloning step3 to have it inserted in out ompC promoter plasmid.
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Because cloning into TA vector will allow us to ohtc equate am of Aequorin-GFP into ompC promoter plasmid. We have to determine the orientation of the Aequorin-GFP insert and choice the rights 5’to3’ orientation.
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The cause of unsuccessful digestion might be the few data after progress A-G PCR, or the concentration was not high enough.
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Dues to late start of our project will we still in our project in trying to insert A-G into PSB1A3 we hope we will successfully construct the plasmid we need and transform into CP919. Finally we will determine the optimal Ca2+ concentrations that can have maximal illuminating of A-G in bacteria to create aim biolight .
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'''TA cloning'''
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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.
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'''PSBlA3'''
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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.
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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.
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Latest revision as of 13:38, 21 October 2009
Discussion
Aequorin-GFP coding Sequence containing plasmid was kindly provide by French scientist() We use PCR to Pst1 site at both ends of Aequorin-GFP coding Sequence. Then we insert Aequorin-GFP lst into TA vector to have easier cloning step3 to have it inserted in out ompC promoter plasmid.
Because cloning into TA vector will allow us to ohtc equate am of Aequorin-GFP into ompC promoter plasmid. We have to determine the orientation of the Aequorin-GFP insert and choice the rights 5’to3’ orientation.
Dues to late start of our project will we still in our project in trying to insert A-G into PSB1A3 we hope we will successfully construct the plasmid we need and transform into CP919. Finally we will determine the optimal Ca2+ concentrations that can have maximal illuminating of A-G in bacteria to create aim biolight .
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