Blunt end cloning with Pfu and EcoRv - (Apr/17/2007 )

I ve done done the following steps in my blunt end cloning work.

Vector - a mammalian expression vector with CMV promoter. It was a gift from my supervisor's friend. The vector has a multiple cloning site (MCS) in the middle of lacZ gene. I cut the vector with EcoRV and gel purified (Qiagen kit) the digested vector prior to ligation.

Insert - Viral DNA (human papillomavirus). A 1, 157 bp fragment was PCR-amplified using Pfu polymerase, and gel purified using Qiagen kit. My pcr band was excellent (clean, sharp band).

Both insert and vector have good 260/280 absorbance ratio(~ 1. 8) when measured using Gene Quant. I performed my ligation using Rapid Ligation Kit (LigaFast) from Promega, using the manufacturer's reccommended protocol.

The results?

Nothing happened. Not a single clone, none what so ever. I was even half expecting that some of the vector might self ligate, given that EcoRV produces blunt end. Even that doesn't happen.

I am at my wit's end here. I ve repeated this experiment for 3 times already and it still doesn't work. Am I missing something here?

Please help me....

May be you ligase is not working. Did you use a positive control?
May be the problem is the competent cell. Did you use a positive control also?

If it is well, try more ligase of longer incubation time.

Try different relations of vector and insert.
With Blunt ends I have good result with: 1:8 ( vector:insert) molar ration.

and to add to this,

is your ligase buffer still okay (that goes off too but not as frequently as the ligase enzyme)
do you still have your DNA just prior to transformation. Can you confirm (by running a gel with a narrow well) that you still have DNA and not lost it somewhere along the way.

Ligate your DNA in a higher concentration, ie same amount of DNA insert and vector (as previously but) in a smaller ligation volume. Incubate overnight, as blunt end ligation can be rather difficult.

Use more insert and vector. (in terms of total DNA)
Use a higher ratio of insert to vector

You may be trashing your DNA during gel purification by exposure to short wave UV. Use 365 nm for a short a time as possible. Also, I would use a "quick ligation" kit, which has PEG containing buffer. These work much better for blunt ligations than the normal buffers.