Phusion kit altering my restriction sites only - (Jan/16/2007 )
I am using the Phusion kit (HF buffer) to amplify genes out of bacteria. I have never had a problem until this last set of constructs I was making. I have engineered restriction sites on the end of my primers so I can cut/clone them later. I do this all the time and again, I have never had a problem. I run out the PCR on a gel and clean it up with a Wizard kit, then I A-tail the product afterward with dATP and Promega Taq so I can pop it into pGEM and cut it back out with the added restriction sites. This time, after sequencing the plasmid with these inserts, my actual gene sequence came back perfect, but the 5' restriction sites were all altered. On three different plasmids, they were XbaI (should have been TCTAGA, it was TTATAGA), SphI (should have been GCATGC, it was TCATGC) and BamHI (should have been GGATCC, it was TCC, the GGA was clipped). Obviously this prevents the cuts that I need. This is the only part of the sequence that is not correct, the gene is perfect and the 3' end restriction site is fine. I have never had this problem before.
The only other thing that is different is that these are ~190bp PCR products. The large PCR products (2700bp) had no problems. I do not want to make the same mistake and have this happen again, but I do not even know what I could be doing. Is it the small PCR product that is a problem? Is the Phusion kit proofreading function chewing back my 5' sites? Is the A-tailing causing a problem? Anyone have trouble with this type of thing before? This kit has always worked wonders for me in the past.
if your product is 190bp, then in my mind chew back is a very good suspect. If chewback is the cuprit, then the elongation time is too long, so the enzyme is allowed to hang around and remove your ends.
I would suggest reducing the extention time drastically, or better yet change to Taq. Since your are sequencing the product (which is very short anyway, thus the likelihood of error is reduced), you should be okay.
Sequence the circularised vector to make sure the restriction sites you assume you have are actually there.
why don't you suspect the oligos therselves?
The sequence is from the complete vector plus PCR insert, that is how I know the restriction sites were altered.
It's not that I don't suspect the oligos. It's just that we always get out oligos from the same place (Operon through Fisher) and have never had a problem. I ordered 12 oligos in that same order and all of the downstream primers were fine, but these 3 upstream primers are the problem. Also, being that it's the Phusion and small products, I was focusing on that first. Lastly, it's more difficult to actually tell if it's the primers themselves. This may be a completely different topic, but how can you verify without a doubt that it's the primer and not something else? Amplify the region and then sequence the PCR product directly?