Trying to cleave out unwanted nucleotide sequence in an insert. - (Jun/03/2008 )
I am trying to PCR clone a cDNA sequence into a HA-tagged vector; however, I ran into a bit of a problem. Upon reviewing the sequence of the insert in the HA-tagged vector, I found an extra 100+ nucleotide sequence that messes up the frame reading. I have tried to check to see if this sequence was in the original vector but all of a sudden the sequencing primers do not work. If I take out this extra nucleotide sequence the insert will be fine. So I need to know some techniques to get rid of this sequence.
One thought I had was to PCR clone a restriction site on both sides of the extra sequence, cut it out and religate. Are there any other faster techniques that I could use get rid of these extra nucleotides?
Thanks a million
Simple! Design primers that are specific to the regions flanking the extra bases you wish to delete. Each primer needs to contain at least 10-15 bases specific to each side flanking the region you want to delete (so each primer is 20-30 bases long). The primers must be page or hplc purified but then you can pcr copy the entire plasmid (aka "round-the-world" pcr). Your primers can be longer if needed but you may wind up with a high Tm and need to play with buffer conditions to get the correct product. I had to increase the pH and drop the concentration of both MgCl2 and KCl in order to get the pcr to work but in one reaction you can delete these bases. I deleted 222 bases in one step.