Hi
I met a trouble in TOPO ligation. I tried to insert a 4.0 kb PCR product (using Pfu) into TOPO vector. I got many both of white colonies and blue colonies. After exation plasmid DNA from 12 white coloies, there were no insert!
Why? I cut off PCR product from modified TAE gel (low EDTA), and purifed using ULTRA-DA KIT. Please tell me why and how?
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milanoj
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Posted 25 June 2002 - 07:38 AM
Yutong,
Many of these heat labile polymerases do not place an overhanging A at the end of the PCR product. Are you using TA TOPO or blunt TOPO if TA the cloning may not work with a Pfu generated amp product. Inubate the purified PCR product with 2.5 units of Taq. This will create an A overhang and your cloning results should improve.
Many of these heat labile polymerases do not place an overhanging A at the end of the PCR product. Are you using TA TOPO or blunt TOPO if TA the cloning may not work with a Pfu generated amp product. Inubate the purified PCR product with 2.5 units of Taq. This will create an A overhang and your cloning results should improve.
