There are three fragments in my ligation, one is the EcoRV(blunt end)-NcoI fragment of plasmid backbone. Another is an oligo(~40bp) containing EcoRV(blunt end) and KpnI site on two side respectively, and the other is pcr fragment cut by KpnI and NcoI. I have tried many many times of ligation, but all the clones i picked out were like the wild type plamid (seemed a bit smaller). Do I need to phosphotase the plasmid backbone? Or should I increase the amount of each fragment and T4-ligase? Please help me...
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#2
george@CASE
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Posted 17 February 2005 - 09:09 AM
how long are you doing the ligation?
Try prolonging it. I have never done a 3-way ligation but someone I know did an overnight ligation at 16C, then the whole of next day at 4C.
what are your molar ratios? I would try to increase the amount of your oligo because of the blunt end ligation.
Try prolonging it. I have never done a 3-way ligation but someone I know did an overnight ligation at 16C, then the whole of next day at 4C.
what are your molar ratios? I would try to increase the amount of your oligo because of the blunt end ligation.
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felixwang
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Posted 18 February 2005 - 06:11 AM
george@CASE, on Feb 17 2005, 10:09 AM, said:
how long are you doing the ligation?
Try prolonging it. I have never done a 3-way ligation but someone I know did an overnight ligation at 16C, then the whole of next day at 4C.
what are your molar ratios? I would try to increase the amount of your oligo because of the blunt end ligation.
Try prolonging it. I have never done a 3-way ligation but someone I know did an overnight ligation at 16C, then the whole of next day at 4C.
what are your molar ratios? I would try to increase the amount of your oligo because of the blunt end ligation.
