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Cloning small PCR fragments

Cloning small PCR fragments - Molecular Cloning Forum

Cloning small PCR fragments - DNA cloning forum. Discuss the cloning of plasmids, vectors, DNA, RNA, cDNA, proteins, and other molecules for expression, sub-cloning, and other methods. Also discuss digestion, ligation, transformation, plasmid preparation, cDNA library screening, and competent cells here.


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Old 02-10-2011, 03:57 PM
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Default Cloning small PCR fragments



What is the best clonning method (TA, blunt end, etc...) for clonning small PCR fragments (20-30bp) to later sequence and blast for species identification?

RT-PCR reaction is generating Ct values for non-specific targets. Need to identify these targets in an effort to design better PCR primers or to explain the non-specific amplification. Sequence homology is the no brainer reason but I need to identify the organism that is homologous to my target sequence.

Please help if possible. Clonning efforts are wearing thin.
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Old 02-14-2012, 07:58 PM
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Default Re: Cloning small PCR fragments

how about random amplification instead so that you get sufficient DNA for sequencing, instead of trying to cloen them?

Last edited by admin; 02-15-2012 at 04:00 AM.
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Old 05-08-2012, 06:30 PM
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Default Re: Cloning small PCR fragments

When I wanted to verify my amplifications from qPCR, I cloned them into ZERO vector (a vector for blunt-end PCR product ligation).
You should run your qPCR reaction in a dense gel (like 2-3%) and cut out your band. Then procede as usual with PCR cloning protocols.
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