Cloning PCR products
s9401501 at COUGAR.VUT.EDU.AU
Sat Oct 25 21:02:29 EST 1997
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.
Centre for Bioprocessing and Food Technology
Phone 61 3 9216 8137
Fax 61 3 9216 8135
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