Wrong sized PCR band following digest & ligation into vector - :( please help... (Jun/25/2006 )

Hi,

I have been double digesting a vector (pFastBac1) and another vector containing my gene of interest, with EcoRI and HindIII.

I digest overnight at 37C, run them on a gel and extract the bands (both are the right size) purify using a gel purification kit (Mo Bio) and then run a small amount on another gel to confirm purity etc and estimate quantities of dna. When i ran both purified samples on the gel they ran at exactly the right size again and no other bands were present...

Then I do my ligation and transformation, and PCR the colonies using vector-specific primers which should result in a band ~200bp bigger than my insert.

Some of the colonies are empty, which is ok because it means incomplete vector digestion, but some of them show an insert of the incorrect size (and none the right size!)...

The first time this happened, almost every colony showed a PCR product approx 100bp bigger than the empty vector control lane, which I am finding it difficult to find an explanation for.

This has happened again today, only this time those showing an insert were approximately 300bp too small to be my gene

Neither time has resulted in any colonies showing a PCR product the right size.

The only explanation i can come up with is contamination, but how could a contamination ligate into a vector with sticky ends? And why wouldn't any of my insert be ligating in, even when I have quite a high insert:vector ratios?

Please help me, this is driving me nuts and I feel very discouraged

I think you have an incomplete vector digestion problem. And only those few products with the single sticky ends are ligating.

ARe you digesting your vector sequentially, with Ecori then hindIII (or vice versa) or are you doing a double (in the same buffer) digest?

What are your numbers of experimental and control tranformants? Do you think you are getting a low number of colonies of vector with insert?

Sorry for all the questions, but it will help determine a quick and easy fix.