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pcr clean-up fail? - (Aug/08/2011 )


I am scratching my head because I have gotten primer-dimers inserted instead of my insert twice in a row. Granted, I am training an undergrad to do all of these steps, but I cannot really figure out where he is going wrong either.

My insert is a 900 bp PCR fragment, which after touch-down PCR shows a bright clean band at the right length.

The process basically goes like this -

1) clean up PCR reaction w/ qiaquick column
2) digest w/ KpnI & SacI overnight
3) clean up w/ qiaquick column
4) ligate into cut & dephosphatased pL4440 vector
5) transform into DH5a

With the two sequential qiaquick PCR cleanup steps, I am astounded that even on the second time around we got primer-dimer and short product inserts that were 20-60 bp long. The qiaquick columns are supposed to wash away anything less than 100 bp, right?

I am going to have him try a gel purification of the band this time, but we always get terrible yield from that protocol (any tips?) compared to PCR cleanup.

Any one else have similar experience with insufficient primer-dimer elimination from PCR cleanups?


after RE digestion you still can see the band?