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#1 rf-cloning troubles » 2nd PCR not working » 2018-03-09 14:30:01

Replies: 1

I know there are a lot of messages on here with the same problem and I've read through many of them and taking the advice on board however I'm still not producing colonies (or correct colonies) after the transformation.
I am trying to insert either a V5 or ollas tag (both 42bp) into my vector and despite the first PCR working fine I don't seem to be able to produce colonies once transformed. I originally tried the V5 tag and when this didn't work I tried an ollas tag instead but have had no luck with either. I have used both the 2 and 3 step PCR and tried with both self made and bought chemo competent cell, to no avail; I have also reduced the amount of digested product I transformed but nothing seems to have helped. If anyone has any suggestions it would be greatly appreciated.


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