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Bisulfite/sequencing-based Methylation Analysi - (Apr/07/2014 )

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I recently designed different set of primers using the online Epidesiger and the Zymo Bisulfite primer seeker. The annealing tempretures came between 50 to 55. I used the EpiTect kit. DNA fragments I am intoregating ranging from 237 to 549 bp. I am trying to make my PCR conditions work. But I got nothing on the gel! Nothing! Not even a smeer. I tried PCR volumes 25 ul and 50 ul with no luck. I am sure I am using 10 uM primers to final concentration 0.8 to 1.0 uM.
I am using the 2X mean green master mix. Other folks in the lab didn't have problem with the use of this master mix. I don't know why the gel has nothing!

-Epigenome1-

Try lowering your extension temperature from 72 to 64 and lengthening your extension time by 50%. Very low GC DNA will not extend at 72 in many PCR reactions. I assume you already have tried lowering the annealing temperature. It's worth trying as low as 48, but I've never gotten anything to work below that. Do NOT believe the calculated Tm for use in your PCR annealing temperature.

-phage434-

Thanks Phage434!

Lots of folks are saying that the EpiTect kit is very reliable. Do I need to worry about the use of the mean green master mix I used? Do you think it may hurts the bisulfited DNA?

I planning to make my own regular master mix I used to use before. How about the cycles number? do you think 35 is good enough?

Thanks

-Epigenome1-

In most cases, 35 cycles are not enough. Are you doing MSP or BSP? If it is for BSP, you can reamplify your first round products.  

-pcrman-

One thing I discovered is that bisulfite conversion of uncut plasmid DNA is very poor. You need to linearlize plasmids if you want to see conversion. Usually this is not an issue, since you care about genomic DNA, not plasmid DNA, but it is something to know about.

I agree that 40 cycles would be prudent in this case.

-phage434-

Thanks folks!

I will be doing bisulfite-sequencing PCR. I am using genomic DNA not plasmid circular DNA. Thanks for the tip though. I will try 40 cycles. The problem is that we do not have a thermocycler that enables a gradient-change of temperatures to make life easier for catching the right annealing temp. So I guess I have to be lucky to get the right Annealing Temp sooer :)  

-Epigenome1-

In most cases, 35 cycles are not enough. Are you doing MSP or BSP? If it is for BSP, you can reamplify your first round products.  

so I clean my PCR products and run another PCR using the same primers?

-Epigenome1-

You don't have to clean your former reaction. Just add a very small amount of the previous reaction as a template to the new one.

-phage434-

Thanks!
What's the advantage for that?

-Epigenome1-

You're effectively doing more cycles on the previous reaction. You don't want all of the reaction components of the previous PCR in your new reaction, just a little template DNA, so you want to dilute the previous reaction. If you dilute by 100x, then an additional 7 cycles will get you back to where you were. From there, you amplify more.

-phage434-
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