PCR - Anomalous mobility of PCR product (Jan/30/2008 )

Hi,

I am trying to amplify the 717 bp gene using PCR. Initially i had problem of getting multiple bands. Latter on i changed the conditions like reduced the primer concentration, increased the annealing temp etc. I got thick PCR band .When I checked by gel, it looks like 600bp band. I used 1kb marker. The only difference I made was the dye used to prepare the sample .I used bromophenol blue to prepare my PCR samples. But Marker came with xylene Cyanol FF dye. Does it make any changes?

PCR condtions used
1. Hot start
2. Denaturation
95C 30 seconds
3.anneling
65C 30 seconds

4.extension
72C 1minute

30 cycles

5. Final extension
72C 10minutes

6. cool 4C


Please give me some suggestions….

pcr

[attachment=4146:PCR6_edi...labelled.JPG]

Thanks
Paul

Hi,

I actually don’t see a huge problem with your PCR product... The band looks pretty OK. If it were up to me, I’d go ahead and use it for cloning (or whatever you want to do with it).
I don’t think you can assess the size of fragments on the gel that accurately – it's more or less an estimate. Anyway, if you want to be sure it’s your desired product, just purify it and cut it with a restriction enzyme. If it gives you bands approximately the size expected, it’s your product, for sure...

I would go for it too. I have been cloning a 2100bp product and it migrates right between my 2kb and 3kb markers.. slightly bigger than I expected but sequence has shown it is correct.