I have been having problems with ligating a 3,200 bp insert into a 5,000 bp binary vector. Digests and amount of DNA are very good. I have been doing in-gel ligation and E.coli (DH5a) transformation (routin protocols) with no success. Any advise??
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#2
John235
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Posted 23 July 2002 - 07:14 PM
You might want to dephosphorylate the vector
dephos enzyme and buffer from Lifetech (GibcoBRL)
dephos enzyme and buffer from Lifetech (GibcoBRL)
#3
cecile
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Posted 04 August 2002 - 05:19 AM
1- you can try to dephosphorylate the vector. Be sure you inactivate the enzyme (5min 85C) before ligation.
2- you could try by PCR. amplify your insert with primers having enzymatic restriction sites to obtain cohesive ends compatible with your vector.
3- your insert is very big. It can be a problem. You can try to do ligation in two times. Cut you insert in two pieces (1,6Kb) and insert one after each other.
2- you could try by PCR. amplify your insert with primers having enzymatic restriction sites to obtain cohesive ends compatible with your vector.
3- your insert is very big. It can be a problem. You can try to do ligation in two times. Cut you insert in two pieces (1,6Kb) and insert one after each other.
