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Old 08-21-2013, 06:16 AM   #1
Junior Member
Location: UK

Join Date: Oct 2010
Posts: 1
Default End-repair at wrong temp

Hi all,

I made a stupid mistake when preparing some libraries for exome capture (Agilent SureSelect XT, Illumina protocol). I accidentally ran my end-repair reactions on my a-addition protocol (37 degrees for 30 mins with heated lid at 50, rather than 20 degrees with no heated lid). I only realised my mistake when I had prepared the a-addition reactions and was loading them in the PCR machine.

Can anyone speculate as to whether or not these reactions will have worked? I think it's the T4 and klenow polymerases that need the lower temperature, but NEB state they have activity at 37 degrees, so I'm hopeful?

I don't have enough DNA to re-start the process, hence why I'm trying to work out what my best options are.

Thanks in advance.
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