Troubles in PCR the converted DNA in promoter region - (Jul/18/2007 )

I was doing the bisulfate sequencing to test the methylation profiling of 5HTT. The interested region is 2026bp in length. I use the Qiagen EpiTect Bisulfite Kit for bisulfite treatment and got the positive converted results. Because the amount of DNA sample is limited for 5HTT,so I consider to perform nested PCR. First round PCR I used the long-PCR kit and got the long-PCR fragment which detected as a sharp band in the agrose gel,then I diluted the PCR product to 200* dilution and used it as the second rond PCR for short fragements. Among the 7 primer pairs, 6 produced sharp bands showed in agrose gel in pre-test experiment,but later only one of them came out stably, and I got good sequence for this fragment.

The initial DNA concentration for Bisulfite are 39-115 ng/ul ,and I use 20ul for bisulfite treatment; the converted DNA concentration are 4-21ng/ul, which I use 4ul DNA in 25ul PCR system in long PCR.

The PCR programs are as following:

long PCR :

PCR system: Each Rx Final Concentration
RNA-free-Water 13.05
LongPCRBuffer with Mg2+ 2.5 1x;2.5mM Mg2+
MgCl2 (25mM) 0
dNTPs(10mM,each) 1.25 500uM of each Dntp
Long Taq(5U/ul) 0.2 2Unit per 50ul reaction
Primers (S) (5uM) 2 0.4uM
Primers (AS) (5uM) 2 0.4uM
DNA 4
Total 25

Program: 93,3min
(93,15sec;51,30sec;68,3min)40cycles
4,hold

using Qiagen Long PCR kit.
Long fragment is 2348bp in lenght .




Nested PCR:

DNA:200* dilution of PCR product from above amplified

PCR system Each Rx Final Conc. Initial Conc.
Water 0
Buffer 1.5
MgCl2 1.5 2.5 25mM
Formamide 0
dNTPs 1.2 0.2 2.5mM
Ampli-taq Gold 0.15 0.05 5U/ul
Primers (S) 0.75 0.25 5uM
Primers (AS) 0.75 0.25 5uM
DNA 9.15
Total 15

Primer pairs: 95,10min
(95,15sec;51,30sec;72,45sec)40cycles
72,10min
4 hold

using ABI Ampli-Taq Gold kit.
Short fragments are 397-512bp in length.


I have stuck in it for about 2 months, are there something wrong in my PCR process? But I have successfully got one fragment which located in exon1 region, why the PCR was so unstable,unrepeatable? Anyone would like to help me with that? Any suggestion will be much appreciated!

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hi sallynie,

it's usually the primers, I can't remember if I have helped you with the design or not.

how were they designed?

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What were the differences between your pretest experiments and your "real"-experiments?

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Hi,nick,

Thanks for reply. I designed the primers by manual and then calculate the Tm by Primer Express. The attached file includes the C-T converted Reference Sequence,primers and PCR protocal.I found today that the primer 3-re is wrong at A which should be Y (

-re: 5’-TAAAACYAAAAAATTACTTAAACCCAAAAA-3’

) Pls see if there are something wrong with it!

Thanks!

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Hi, Krümelmonster, the differences between my pretest experiments and "real"-experiments mainly are: Firstly,when I used different block(Hybrid) for real-experiments --- I could not get the PCR prod;then I used the same block (ABI 9700) as the pretest experiments --- I still could not get the PCR prod; after that,I used the same block(Hybrid) do the gradient PCR --- unfortuately,PCR still not work. I have an headache on them now! The reagents are definitely good.
Perhaps the only way I can do now is redesign the primers.

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Try a lower extension temperature. 64C instead of 68 or 72, which you are currently using.

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Hi,I lowered the extension temperature to 64C for 4 primer pairs,but still got no band. Then I will change the primers.Thanks for all reply!

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