BSP PCR standardization...help - (Aug/26/2009 )

Hi,

I am having trouble optimizing the PCR reaction for my bisulfite treated DNA. Bisulfite treatment and clean up of human genomic DNA was done using Qiagen Bisulfite Epitech kit. After that i did a touchdown PCR with annealing temperature ranging from 62C to 54C ( total number of cycles 35, 2C step down) but got no band on agarose.

Tm of forward primer and reverse primers are - 57C and 60.4C respectively.

Queries:
1) should I increase the annealing temp and by how much.
2) Is 35 cylcles sufficient.
3) I read that for bisulfite treated DNA PCR,final extension is done at 65C. Is it necessary to do so.

If any other suggestions...Please let me knw.

Regards,
PKS

PKS on Aug 26 2009, 10:56 AM said:

It's always good to run a temperature gradient for PCR optimization. The number of cycles strongly depends on your DNA input amount. I use 10 ng per PCR and run 45 cycles. And I use an extension temp of 72°C...

Hope that helps...

Best,

MoB

how did you design your primers? this is absolutely critical to getting a PCR amplicon from bisulfite converted DNA

methylnick on Sep 1 2009, 07:47 PM said:

Primers were designed using 'methprimer'.

I have also tried PCR at annealing temperatures of 57C/40secs (40 cycles) and 63C/40secs (40 cycles). Only a faint smear was observed on 2% agarose....

methprimer isn't very good at designing primers that amplify converted DNA sequences, the best primers always end at the 3' from a converted c (in the primer it's a T).

The best software I know of that conforms to this rule is methyl primer express from ABI


Nick

Hi, I´m new here. This is my first post. I´m trying to analyze the methylation status of a promoter too. I extract DNA from several rat tissues using the phenol-chlorophorm method. Then I use the DNA Imprint Modification Kit from Sigma with 1 microgram of DNA. After that I do a MSP using primers designed by Methyl Primer Express Software. In my first PCR didn´t get any specific band (I´m looking for a 244pb band) with any of the primers of the set (primers for methylated sequences and primers for unmethylated sequences) . Then I change the reaction conditions (changing the Mg concetration, annealing temperature and decreasing primers concentration to 0.2 micromolar. Doing that I get a band in some samples and I decide to sequence that products. But the sequence I get isn´t the one I looking for. Then I thought that the primers were not specific for my promoter, that´s why I get a plasmid with my sequence to have I positive control. After that I treated the plasmid with the same Imprint Kit from Sigma with the same protocol that I used for the genomic DNA. I run a MSP with my plasmid, and I get a product with both set of primers. But when I try with the genomic DNA samples I don´t get a band of the size I´m looking for.
Any suggestion...????
Thanks....

I strongly feel that your touchdown should be 0.8C / 0.5C step down. So from 62 to 54, you need 10 / 16 cycles. Run 54 for another 20 - 25 cycles.

Also it depends on your annealing time. Preferably 40s for ~ 800bps and another 50s for extension time.
Try and let us know the outcome.

PKS on Aug 26 2009, 04:56 PM said:

rafel61 on Oct 6 2009, 09:27 AM said:

Hi Rafael,

you can try more genomic DNA template in your PCR, plasmid is less complex than genomic DNA and therefore there are far more copies of your sequence of interest in your plasmid compared to your genomic DNA.

Nick

Hi...Yes you are right...I will try to use more DNA, and I will change my bisulfite modification procedure. I believe is better to do the bisulfite conversion without kit. Because I think the Sigma Kit isn´t working at all...

methylnick on Oct 7 2009, 04:04 AM said: