PCR trouble - (Oct/03/2005 )

Hi,

As I said before I finally amplified 1970(very weak) but with other 500,700,750 bps.I purified 1970 from gel and did TA cloning. I selected white colonies and did colony pcr of selected 10 white colonies and run it on gel. but I saw again 500 bp with some colonies and 700 bp with others!!!and this time I used M13 forward and reverse primers for colony pcr!! so if my primers are the problem how is it possible to see again 500, 700 bps in colonies that I transformed with 1970 including construct??:(and also I used M13 not my primers?

I attach gel photos of both amplification of 1970 and colony pcr results.

first photo is 1970 amplification.There,11970 is the top band.
second photo is the colony pcr results.


any idea will be appreciated !!!!!

thanks...

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The 1970 bp fragment you purified from the gel carried over with it some of the 500 bp and 700 bp products. These will clone much more easily than will your 1970 bp fragment, and thus were over represented in your TA clones.

I would think about the following:

• Select new primers or devise new PCR conditions (perhaps using something like Invitrogen's PCRx enhancer kit) so that the band you're interested in is at least the majority product.
• Run your original PCR out on a long-bed gel (25 cm) very slowly (like at 40 V overnight in TAE) to achieve better purification.
• Digest the TA clones, to confirm they are clones of the smaller fragment (although they must be, 'cause you used vector-borne primers for the screen).
• Sequence one of the 500 bp and one of the 700 bp TA clones to see where these smaller products are coming from. This may help you in designing better primers, and may be biologically interesting in itself...
• Pick and screen more TA clones -- the correct clone may be there somewhere...

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Hi HomeBrew,

Thanks a lot for your advices:)

Actually I tried hotstart taq for enhanced pcr but nothing changed.I am using GC-melt in all pcr reactions because template is %56 gc rich.

I will continue to screen other 1970 colonies and sequence 500 and 700 bp fragments at first...

Thanks again...

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Good luck! You might also try PCR'ing your product up from the gel purified first reaction -- I assume the correct template is actually there in higher proportion than the small fragments, but the small fragments are cloning easier (and the resulting plasmids are also smaller than your correct one, and thus will transform easier -- skewing your colony population even further).

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Yes I tried that one .Gel purify the band and did another PCR from it but could not see anything except smaller fragments.But I used a different PCR program for it.So now I can try the same program that I used when I saw the1970 band with this purified fragment.I think you will say why did you change the program, are you crazy?:)Hmm at the beginning I used 60 annealing temp.and saw nothing.then I tried touchdown from 62 to 60 and saw the band but weak and I thought if I use 62 annealing temp only I would have a specific, brighter band but again saw nothing:)and then I thought I can try TA cloning with that weak band:( So now I will try again touchdown.

Thank you a lot for the answers...:)See you.

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Dear Katanin,

Regarding to the gradient function, you need to have a gradient cycler (eppendorf).
This allow you to set different annealing temperature to each well. This will help you to find out the best annealing temperature in one run.

Secondly, judging from you problem, I think your primer is not specific. The primer
instate of bind to the correct place to generate you a fragment of 1970, it more bind to several places as well. Thus you get these 500, 700 and 750 band constantly.

So I would like to suggest you to redisign your primer, and I will save your trouble.

Best regard

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Hi Hadrian,

Yes I think so and after I do my last PCR with that primer (last chance:))I will redesign my primer.

Thank you for the answer:)

See you...

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