10 replies to this topic

[labtechie]

I am able to very successfully get small (150-200 bp) products using a nested PCR strategy on bisulfite modified DNA; I do not see products from the larger primers but they are certainly there since the nest works so well.  I'd love if I could sequence those larger products (400-500 bp).  Does anyone have any hints to get a visible PCR product?  Can I just ramp up the cycles?  (I do 35 cycles and an MgCl2 concentration of 3 mM)  Before you tell me this is just a normal PCR problem, you should know that most papers specify that a nest is necessary, one should not expect to see the larger product, and larger products after modification are fragile and difficult to get.

Thanks!!

labtechie

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[methylnick]

Hey Labtechie,

I have been able to successfully amplify and sequence 1kb from bisulfite treated DNA.

A nested PCR strategy is the best option. I typically design a hemi-nested primer set that is, I pick a primer pair (A and that amplifies around 900-1.2kb in length and then a third primer © within the pair.

So this would require two rounds of PCR amplification. In the first round of PCR I would use Primer A and B, take 1uL of this into a second round of PCR with primer C and either A or B depending on how you have designed things.

If this fails a magnesium titration will do the trick. Again with the hemi nested PCR I would titrate MgCl2 at 0.5mM final concentration intervals.

so I typically do 0.5, 1, 1.5, 2, 2.5 and 3mM.

good luck techie!

Nick

[pcrman]

Hi Nick,

I am interested in knowing what cycle number you use for the first and second round PCR and how long you treat your DNA (overnight or shorter).

Thank you.

[methylnick]

pcrman, on Jan 27 2005, 04:25 PM, said:

I am interested in knowing what cycle number you use for the first and second round PCR and how long you treat your DNA (overnight or shorter).

Hi pcrman,

the cycle number I use is 30 cycles for each round of PCR.

Here are my cycling conditions:

95C 4 minutes denaturation followed by 5 cycles of :

95C 30sec (denaturation)
annealing for 90sec (annealing temperature set at 2 degrees below calculated Tm)
72C for 120sec extension.

and then 25 cycles of:

95C 30sec (denaturation)
annealing for 90sec (annealing temperature set at 2 degrees below calculated Tm)
72C for 90sec extension.

Final extension period at 72C for 4 minutes and then a hold at 4C.

This is repeated for the second round of PCR.

I am unsure how you get the degrees symbol so I have omitted it.

The bisulfite treatment was overnight at 50C in the dark, and desalted with Wizard Columns.....

I have also used Human Genetic Signatures' MethylEasy Kit with the same success. The kit is certainly much easier than the conventional method.

I have used AmpliTaq from Applied biosystems and PCR mastermix from Promega and they both perform very well with bisulfite PCR in my hands.

Cheers

Nick  

Edited by methylnick, 27 January 2005 - 04:40 PM.

[pcrman]

Thanks for sharing. Very impressive.

[miner]

Hi methylnick：
  I  did nested PCR and have been successfully amplify. But ，it is very unfortunate that the sequence is not I want to. The length
is also not I have designed.  What I should do? how to avoid unspecific  amplify. Thank you very much.

[methylnick]

are you sure your primers are designed to the right sequence? could there be other repeat-like sequences that your priemrs are targeting?

you could also try increasing your Tm

[Christina]

methylnick, on Jun 26 2009, 10:45 PM, said:

are you sure your primers are designed to the right sequence? could there be other repeat-like sequences that your priemrs are targeting?

you could also try increasing your Tm

Hi methylnick
I design many primers,but it did't work at all,smear is often.I think my primer is good,what should I do?how to calculate the Tm ?And which software do you prefer to designing primer?
Thanks.

[Laver]

Hi, I think the primer design ought to be the critical step when you do the PCR. You need to avoid use too many degenerate base for the primer. I used up to 4 sites for one of my primer and it work pretty good.
I usually use two pairs of primers to do the nesting PCR. The first round PCR should be in a higher strigent condition, i.e. use higher annealing temp and ramp at -.1 or -.2 degree per cycle. I run it about 30-35 cycles. The second round can be less strigent.
I usually did many different PCR in one block. There's some variations of Tm in each primer pairs but PCRs worked really good. Only very few primer pairs did not work. So I guess you ought to work more on primer design rather than titrate the MgCl2.
BTW, I usually amplify 400-700bp. I did succeed a 1.2kb products. But larger ones are indeed harder and sometimes not very necessary.

[Lilip]

Nick and others

I was thinking that to performe a nested on BSP, the second primer would go into the CG sites. Do you design primers with an interval to the second primer out of the CG region?

Why do you make the extension step longer in the first round? Annealing step of 90s is not too much?


I performed the PCR  ( 700bp) and than purified my band from the gel an than a second PCR from the purified product ( with the same primers) and a got the same nonspecific (250bp) band of the first PCR!!!! Why was that? My primers had annealed in my specific band? I got the same result with methylated and non-methylated DNAs controls.

[Tanya C]

methylnick, on 27 January 2005 - 04:39 PM, said:

pcrman, on Jan 27 2005, 04:25 PM, said:

I am interested in knowing what cycle number you use for the first and second round PCR and how long you treat your DNA (overnight or shorter).

Hi pcrman,

the cycle number I use is 30 cycles for each round of PCR.

Here are my cycling conditions:

95C 4 minutes denaturation followed by 5 cycles of :

95C 30sec (denaturation)
annealing for 90sec (annealing temperature set at 2 degrees below calculated Tm)
72C for 120sec extension.

and then 25 cycles of:

95C 30sec (denaturation)
annealing for 90sec (annealing temperature set at 2 degrees below calculated Tm)
72C for 90sec extension.

Final extension period at 72C for 4 minutes and then a hold at 4C.

This is repeated for the second round of PCR.

I am unsure how you get the degrees symbol so I have omitted it.

The bisulfite treatment was overnight at 50C in the dark, and desalted with Wizard Columns.....

I have also used Human Genetic Signatures' MethylEasy Kit with the same success. The kit is certainly much easier than the conventional method.

I have used AmpliTaq from Applied biosystems and PCR mastermix from Promega and they both perform very well with bisulfite PCR in my hands.

Cheers

Nick  

Hi methylnickwe've been struggling in one BS PCR for a long time, and Thanks very much for your posted PCR condition, we've got a band, not so sharp or bright, but we're really grateful,comparing nothing before! Now we're thinking about increasing the cycle or trying other pairs of nest PCR. Are there other factors should I work on? I've done the magnesium titration, and have found our optimal con.(1.5mM). Any suggestion would be much helpful.Thanks in advance!Tanya