Blunt cloning - No insert, despite lots of colonies - (May/07/2013 )

I'm trying to clone PCR fragments into a blunt end vector backbone.

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:

1. PCR
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.

If you clone into a TA vector you can later digest and subclone. What's the problem? At least it's more efficient than RE digestion of the ends of your pcr product. They don't digest very well, or the concentration of your pcr product after purification could be low. And finally, prepare fresh agar plates if your plasmid is amp res.

I don't want to use TA for it being a. expensive and b. subcloning is not feasible for most of my inserts as they contain common RE sites. I don't do any digestion of my PCR products, as I do blunt cloning. In fact I sequenced a few clones of the PCR backbones and found that the problem is ligation of the unphosphorylated PCR backbone, hence the PNK is not fully inactivated by the NEB recommended treatment for 20 minutes at 65 C. I'll try to phosphorylate the primers instead.

I see. Have you checked pJET? Maybe it doesn't have common RE sites.