Hi, I am linearizing a 8kb lenti puro vector with PCR prior to Gibson assembly. I have precious few reactions in my Gibson kit so I don't want to use something until I'm sure what it is. Currently I am preoccupied with the mystery of why the PCR seems to work very well (get expected band) only when I use the tubes that melt in the thermo cycler, but when I run the same reaction with tube strips that never melt, I barely see any band (mostly a smear).

Reaction - 51.5uL total

Template Plasmid Dna 43ng/uL - 1uL

mol. bio. H2O - 35uL

DMSO (has been freeze-thawed) - 2.5uL

Q5 5X reaction buffer - 10uL

Q5 Polymerase - 1uL

10uM Primers (F/R mixed) - 1uL

According to eurofins genomic and snapgene my primer Tm are 65-66 C.

My Program

95C - 5min

95C - 20 sec

61C - 20 sec

72C - 3 min

25X cycles from steps 2-4

72C - 5 min

4C - infinity

I am currently running an experiment to see if splitting the reaction volume in 2 parts or increasing the Ta to 67C will help.

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