Problem is as follows: I get lots of colonies, but none of them seem to have the insert. However, my 'empty ligation' control plates show very few colonies.
I prepared vectors in 3 different ways:
2. Blunt digestion (EcoRV, over night) + FastAP
3. Sticky end digestion (NotI-HF), T4 Polymerase blunting, FastAP
Inserts are produced by PCR (Takara PrimeStar GXL), gel purified, T4 PNK phosphorylated.
Both FastAP and PNK are heat inactived at 65C for 20 minutes.
Ligation is carried out over night at 16C with 2 uL T4 ligase in 20 uL. 10 uL are transformed into TOP10. I do a control where I add the same amount of vector but no insert.
My ligations with insert yield lots of colonies (100s), whereas the negative control for all three vectors yields little colonies (10s or below), with the PCR as expected giving the fewest background colonies.
When I screen the colonies by RE digest after Miniprep (Column kit) everything looks like the negative control despite the inserts having 2 - 3 sites of the respective enzyme in their sequence.
I do use quite large constructs, the insert range from 4kb to 9.5kb, the backbone is either 5.7kb or 7.7kb.
I don't understand how I can get many-fold more colonies in the ligation, yet don't see any insert. Especially since one backbone is an unphosphorylated, gel-purified PCR backbone, which can't recircularize at all (I understand the background is coming from residual plasmid template used in the original PCR). The only idea I have is that somehow the heat inactivation is not working and the PNK would phosphorylate my backbones. But for both the enzyme and ATP to survive the (manufacturers recommended) time at 65 C seems very unlikely to me.
Any ideas are greatly appreciated. Unfortunately TA cloning is not an option since I need specific backbones.
Edited by rxzlmn, 06 May 2013 - 10:51 PM.