End-polishing and ligation

Alan Lee ywlee at hkucc.hku.hk
Mon Aug 25 07:47:55 EST 1997


Hello!
Please help me out! I have subcloned a PCR fragment into pBluescript II
KS vector and now want to reduce the size of the subcloned fragment by
1kb. The approach was to digest the plasmid with AccI (1 site in the
subcloned fragment & 1 site in the multiple cloning site of vector) to
release the unwanted trunk and religate the plasmid. Since the AccI cut
generated incompatible ends, the AccI-digested plasmid (3.6 kb) was
blunted and self-ligated in a single tube reaction as follows:
	AccI-digested plasmid (gel purified): 130ng
	10x NEBs T4 ligase buffer <with 10 mM ATP): 1ul
	4 dNTPs: 100uM
	NEBs T4 ligase: 1ul
	NEBs T4 polymerase: 3U
The reaction volume was 10ul, room temperature for 16 hrs.
I have tried several time with this protocol without success.
The competent cells were fine and all reagents used were
fresh. I don't know what has gone wrong. Please kindly advice me on
this issue.
Best regards.
Alan
e-mail: YWLEE at HKUCC.HKU.HK



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