Homo & heterodimer Problem - (Jul/25/2006 )

Hello everybody,

I have a primer pair which has Average Tm about 58 degree and no significant dimer formation according to IDT tools Oligo analyzer. But then for the purpose of cloning I need to add a NotI restriction site (5'-GCGGCCGC-3') at the 5' end of my primers and in this case I will get both hetero dimer and homodimer.Since the NotI restriction site has only GC the Tm also will be pretty high and they will form quiet stable structure.I can't use any other enzyme becoz the vector I am going to clone it into has only NotI and AleI in the region where I want to insert my sequence.

I forgot to tell u the size of my amplicon is 5600bp.

IS there any solution for this problem..CAn I amplify this region by using primer without the restristion site and then add the restriction site later..If so can somebody tell me how to do it..
I am stuck with this problem and don't know what to do?

Any help would be great..
thanku
Sminim

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Presuming your 3' ends of your primers are unique, and you don't have complementary sequences at the 5' and 3' ends of each primer (which you have already said you are confident of), the fact that you get a bit of homodimerisation of your primers is of nuisance value only, because it is only going to reduce your efficiency. If the 3' bases were complementary, you'd be out of luck, and would have to resynthesise.

Three suggestions to improve the amplification.

1. Try a touchdown PCR. At the upper end of the temperature range, your primers should be (mostly) monomeric, which will give you best efficiency of bindingto the template. By the time the annealing temp has dropped to the point where homodimers are forming, you should have sufficient primer:amplicon duplex that the efficiency won't drop too much. Digest the product, gel purify and live a happy cloning life.

2. Try adding bit of DMSO or betaine to disrupt the primer homodimer. It will probably be a bit tricky to judge how much, because the DMSO will also be disrupting the primer:template duplex. As a slight twist, you could do maybe 5 or 10 cycles with DMSO, transfer 1ul to another reaction mix without DMSO, and continue the reaction. Digest the product, gel purify and live a happy cloning life.

3. Synthesise new primers without Not1 sites, and also synthesise Not1 linkers (you could even make these with the sticky end, so you didn't need to digest the insert with Not1 at all). Amplify the insert, blunt-end ligate the linkers, then digest the product, gel purify and live ... well, you can guess the rest of the sentence.

Good luck...

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thanku I will try doing what u have suggested.
In the DMSO step ,you have suggested me to add 1 ul of rxn mixture without DMSO to the reaction containing DMSO after 10 cycle. But I am unable to understand whether 1ul of reaction mixture without DMSO will make any difference.Do u mean 10ul??

thanku and hope I have a happy cloning life
Sminim

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A couple of points.
I recommended 1 ul only because PCR is one of those expts where "less is more" rules, most of the time. You could add more from the first reaction, I suppose. Whatever give you the result you're after, I reckon!
Also, make sure the first reaction has DMSO, and the second one doesn't (you message got the two switched around).

Happy cloning!

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Very very simple solution here... just add a few nucleotides on the 5'end of the NotI restriction sequence of your primers. You need to add some anyway because enzymes don't work well right at the sequence ends, see
http://www.neb.com/nebecomm/tech_reference...nucleotides.asp

The nucleotides you add can be any sequence, so you can make sure that there won't be any dimers.

LeserattePD

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Hello Swanny and LeserattePD,

The touch down protocol which Swanny suggested worked well and I could get the PCR to work. I did the initial annealing at 60 degrees for 10 cycles and then decreased 0.5 degree every cycle for 15 cycles and then amplified at the lowest annealing temp for an extra 10 cycle ( this helped to increase the yeild..
As LeserattePD has suggested I have addeda few extra bases in my primers for my next set of PCRs.
Thanku both for ur valuable suggestion..
hope my happy cloning life continues

thanku
Sminim

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