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Problems with crossover PCR - (Apr/15/2013 )

Hey again everyone,
I have a feeling I am going to be posting a lot on here. This website has become a very valuable resource for me lately.

Well, the recent snag is that I am trying to do crossover PCR to delete a gene. I have both the 3' and 5' pieces. I amplified the 5' piece with ease..first try. The 3', however, was a bit complicated...the Tm for the SOEing forward and Rev was calculated to be 68 C but I ended up having to increase the actual temp in the thermocycler to 72 C due to non-specific binding. Well, I got the 3' eventually and now I am at the 2nd PCR where I am adding the two pieces together. This is where it gets odd. The 5' piece is 1.2 kb and the 3' is 1.3 kb and after I run the PCR, and analyze on the agarose I keep getting a huge band at 1.2-1.3 kb...I get a very faint band around 2.5 kb..which is where the band should be. The annealing temp for the for and rev is 68 C so I have ran the PCR at increasing temps from 63-67 C and as I increase the temp the faint band at 2.5 kb gets fainter and fainter as the temp increases.

I have uploaded a picture.

Lane 1 is Molecular weight marker- 2 log ladder from NEB
Lane 2 and 3 are the SOEing PCR after the second round. I do everything in doubles in case I screw one up...they both were the same conditions...

I run 25 uL reactions initially until I get the kinks worked out. For the master mix I add:

18 uL ddH2O
0.125 uL taq
0.5 uL forward primer
0.5 uL reverse primer
1 uL DMSO--I work with Pseudomonas which is GC rich
0.5 uL dNTP
2.5 uL Taq Buffer

and then I add 1 uL of each piece (5' and 3' after gel extraction and running on an agarose gel to make sure I didn't lose it)...

Do you have any suggestions as to what I can do to get the correct amplification?
Attached Image


Have you tried gel extracting and cloning the 2.5kb band? You can do this "in gel" if you like (protocol here) You could also purify and sequence, though gel extraction typically gives quite low yields.

Also, have a look at this protocol: Note the info about polymerases. Taq is typically not very good for cloning stuff as it is quite error prone and only really good for short products (less than 1kb is best).


I thought about gel extracting it the 2.5 kb band but I was afraid of losing a good bit of the SOEing product and with the low amount already there I was afraid of losing all of it. I guess it's worth a shot anyway. I can try to gel extract it and subclone it.
I guess I'm more concerned with what could cause the band at 1.3 kb? It looks like each of the fragments are amplifying without annealing to one kind of looks like there is a space in the middle where there are two bands...maybe I'm just seeing things...