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sequential PCR and purification before TA cloning - (May/11/2008 )

Hi all,
The following is what I'm currently doing:
1. I amplify a 3.5 kb using iProof (BIO-RAD) that will produce blunt end product
2. I gel-purify the product using Qiagen because it always produce nonspecific fragments
3. I add A (adenine) using Taq polymerase into the gel-purified product
4. My previous work, I directly use half of the amount in the number 3 mixture to use it in TA ligation into pCR2.1 without purifying the number 3 mixture. For this I never got positive transformants

Thereafter, I purify (direct PCR purification, not gel purification using) solution in number 3, and use it for TA cloning. But the problem is the amount of product was reduced greatly (there was no signal in gel), so I couldn't continue to clone it.

I really need advice, help, etc... thanks in advance


anyway, I can do number 3 first before number 2, so I add A to all PCR products there then gel-purify it