Problems with PCR for GATEWAY - I'd get rid of 100-150bp unspecific band!!! (Nov/15/2006 )

The situation follows...I have to clone a cDNA fragment (2700bp) using attB sites Gateway compatible. I optimized the reaction obtaining a very very good band for 2700 bp BUT I always have a band around 100-150 bp too...I mean ALWAYS!
Any suggestions?

Thanks a lot in advance!

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may be there are primer dimers or something. anyway if you've read the manual, you would have found this remark

so do an agarose gel, cut out the band, elute the DNA, do the BP-reaction ... Bingo!

sometimes, it's not only about optimisation of the PCR

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Thanks a lot Kersten,

I tried already to cut the band...it doesn't work. Actually I had colonies but...the small fragment was inside!!! Quite tricky isn't it?

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please describe your procedure as exactly as possible. I never heard that that is possible

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Ok, I did the PCR with attB sites-specific primers. There were 2 bands on gel: the one expected which was very very clear and the another one (not as clear as the first one) around 100-150 Bp. Then I ran on gel (Syber-safe) 20 microliters of the reaction, I cut the first band with a scalpel, I extracted the band with QIAquick Gel Extraction Kit Protocol, I did the BP reaction using 2 microliters of the eluted band on 10 total volume of the reaction. I had 4 colonies out of 4 plates (!!) then I did the colony PCR with M13 primers (I used a pDONR P2R-P3) and with specific primers. With specific primers I had nothing but the expected band for M13 primers amplification without insertion in the plasmid is around 250 but I had a band around 400bp (= 250 + 150).

I have a doubt only with extracted fragment quality with was bad (the typical DNA peak was shifted on the left of the graph (near 230). I used nano-drop to check it.

Regards

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hi, sorry for the late reply, I was ill.

did you try to just go with the precipitation with MgCl/PEG? the band could be primer dimers, and eventually will not precipitate. you could precipitate and recheck on a gel wether the band has disappeared.
manual:

did you have a control for transformation efficency? if yes, and you know your cells transform well, then I would say the BP reaction was bad, which could be because of the extraction from the gel.
manual:


May be that helps?

regards

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