Cloning PCR products

[Matthew Knight]

Hi all,

I am currently trying to clone a 0.65 PCR prod into a 7.2kb vector
with great difficulty. I am using two different enzymes and have tried
several different ligation ratios of 1:1, 3 insert: 1 vector and 5 insert
: 1 vector. 

I am digesting my PCR product and I am sure it is cutting due to a
size differnece on agarose gel and then I am heat inactivating and ethanol
precipitating the DNA.

I am gel purifying my vector with a silica matrix. My control
transformation is working fine into E.coli TG2.

Does anyone have any suggestions on ligation ratios and DNA
concentrations. For example I am ligating approximately 30-50ng vector
with between 5-15ng insert.

Thanks in advance.

Cheers
Matthew Knight
Centre for Bioprocessing and Food Technology
Victoria University
Phone 61 3 9216 8137
Fax   61 3 9216 8135