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I have amplified my insert DNA (400 bp) by PCR. Then double digested it with Bam HI & Xho I (from NEB) for 4 hrs. Same with vector (pET vector from Novagen. no dephosphorylation was done on vector). I ligated them with T4 DNa ligase (Takara) for 1 hr at 37 deg.cel.
I got very nice colonies in DH5a competent cells (15~20 in number). I isolated the plasmid+insert by mini-prep (Bioneer kit).
I used that mini-prep elution for colony PCR, which shows presence of insert but the sequencing shows the presence of vector with both restriction sites present as it is. I have repeated the whole process twice, still same results.
If the PCR shows amplification of Insert, that means my ligation & transformation step was right.
But the sequencing shows insert absent.
Why does this happens??
What causes PCR to show insert or sequenceing to show insert absent??
This is driving me crazy. I am all out of ideas
Any advice/ suggestion will be grately appreciated.
Edited by Chinar, 27 October 2010 - 01:17 AM.