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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.
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.