t****t
发帖数: 610 | 1 I'm using ZymoResearch EZ DNA kit to convert genomic DNA. Unmethylated and
methylated DNA controls from Milipore were converted together with my
samples.
I use qPCR based method to check methylation. Basically, if it's methylated,
the Tm will be higher that unmethylated DNA due to less C->T conversion.
My experiments were fine until recently. I always found all my samples,
control DNA (methy+unmethy) had the same Tm, which is even lower than (~2
degrees) supposed unmethylated DNA Tm. It looked that everything was "over-
converted".
Can anyone give me some suggestions? | s******s
发帖数: 13035 | 2 没做过tm，要么你seq以下看看？
methylated,
【在 t****t 的大作中提到】
: I'm using ZymoResearch EZ DNA kit to convert genomic DNA. Unmethylated and
: methylated DNA controls from Milipore were converted together with my
: samples.
: I use qPCR based method to check methylation. Basically, if it's methylated,
: the Tm will be higher that unmethylated DNA due to less C->T conversion.
: My experiments were fine until recently. I always found all my samples,
: control DNA (methy+unmethy) had the same Tm, which is even lower than (~2
: degrees) supposed unmethylated DNA Tm. It looked that everything was "over-
: converted".
: Can anyone give me some suggestions?
| R****n
发帖数: 708 | 3 you are talking about Methylation Specific-HRM, I assume. Althought you are
using qPCR system, you didn't use a beckman probe to identify the sequence
change. So this is not a qPCR method.
ZymoResearch's kit should be fine. We used their kit a lot. There are some
commercial M or UM DNA standard which have been converted. You can use them
to test your primers.
How do you design your primers? flanking any CpG sites?
If you didn't change primer, I guess that you may have PCR product
contamination in your standard and DNA sample. This is a very possible
explanation. | t****t
发帖数: 610 | 4 Thanks. It's Methylation Specific-HRM. I'm studying the same CpG islands,
so primers are all the same.
Primers do flank CpG sites.My primers work.
Even I have PCR product contamination, how it is possible to get a Tm lower
than unmethylated control? I have PCR controls, which are already converted
standard DNA (methy and unmethy). They were fine with the right Tms.
Also, although I'm using the same reagents (kit, buffer,...), last week
there was once that my results were fine. Really got confused.
Is there any other recommended kit? | R****n
发帖数: 708 | 5 If memory serves me correctly, kits usually use either beads or spin column
to capture the modified DNA strands.
Is your DNA come from cell or clinical sample? You can wash modified DNA
with TE buffer or your kit buffer a couples of more times to make sure
templates clean. Sometime I did have strange melting curves from clinical samples due to inpurities from biofluids. Or you can dilute your templates to see if there is any change.Even sub-nanogram concentration should work.
From your description, sounds like a primer dimmer or nonspecific PCR
product. I was using HRM kits from qiagen,roche and ABI. Qiagen works best.
Remeber HRM dye is high concentration salt.
Cheers,
Rinman |
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