bisulphite treatment by kit - need information about it and successfull in DNA treatment (Nov/09/2006 )
nothing but praise for the kit, you will be surprised by the amount of DNA you get back for assays, I think it blows Zymo out of the water in that respect.
The guys at HGS are very helpful and full of knowledge as well.
Double thumbs up from me
N
Any opinions on the MethylEasy kit anyone?
nothing but praise for the kit, you will be surprised by the amount of DNA you get back for assays, I think it blows Zymo out of the water in that respect.
The guys at HGS are very helpful and full of knowledge as well.
Double thumbs up from me
N
have you information about EpiTectbisulfite Kit by qiagen?
Hi full-methy,
I work with the Epitect Kit from Qiagen and it works really good in my hands. When treating small amounts of DNA use the carrier RNA provided; that really blows up your yield.
Don't know if it is more expensive than the Methyleasy Kit? 
Krümel
Hello!!
I'm a new memeber and I've got some problems with the Cpgenome DNA modification Kit from Chemicon. If someone has already use it please can you help me because I've no product at the arrival. Thanks a lot!
Ilofleur
Hi Ilofleur,
(1) yes, we used Chemicon Kit, too, but had better results with Qiagen Kit and Methyleasy.
(2) How do you determine your DNA yield? Bisulfite treated DNA is single-stranded and can't be properly measured by light absorption... Did you try a PCR or Digestion after bisulfit modification of your sample DNA?
Krümel
(1) yes, we used Chemicon Kit, too, but had better results with Qiagen Kit and Methyleasy.
(2) How do you determine your DNA yield? Bisulfite treated DNA is single-stranded and can't be properly measured by light absorption... Did you try a PCR or Digestion after bisulfit modification of your sample DNA?
Krümel
Hi Krümelmonster,
I have tried a PCR after bisulfite modification, but I've got no bands in any lane. I wonder if i've got really dna after the modofication, is there a way to know how much dna i've got, before the pcr?
Thank you for your answer.
From memory, the Chemicon kit does not come with validated primers as a positive control for bisulfite conversion,
The methyleasy kit does and it works well, included is a sample of bisulfite convert DNA also.
There really isn't any way of determining you have a template or not as there is not enough to run on a gel, and as a poster said before it's single-stranded, furthermore, it's almost a DNA-RNA "hybrid" molecule whereby there are uracil nucleotides as well as all the others, so I am sure that would affect a spectrophotometer reading somewhat.
Do you have a primer set that is known to work on bisulfite treated samples?
Nick
The methyleasy kit does and it works well, included is a sample of bisulfite convert DNA also.
There really isn't any way of determining you have a template or not as there is not enough to run on a gel, and as a poster said before it's single-stranded, furthermore, it's almost a DNA-RNA "hybrid" molecule whereby there are uracil nucleotides as well as all the others, so I am sure that would affect a spectrophotometer reading somewhat.
Do you have a primer set that is known to work on bisulfite treated samples?
Nick
Hi Nick!
Yes indeed, I've got a primer set that is know to work on bisulfite treated samples. It comes from an article I've read. But on a gel i've got no pcr product. It's a little bit disturbing don't you think?
I wonder if the starting amount of dna is not degraded during the bisulfite trnsformation.
In your experiment what did you do after bisulfite treatment?
Do you think the DNA must be digested before bisulfite transformation or after?
Please help me!
(1) yes, we used Chemicon Kit, too, but had better results with Qiagen Kit and Methyleasy.
(2) How do you determine your DNA yield? Bisulfite treated DNA is single-stranded and can't be properly measured by light absorption... Did you try a PCR or Digestion after bisulfit modification of your sample DNA?
Krümel
hi Krümel
which is the amount of DNA that you treated by Qiagen Kit ?
Hi Full-methy,
I have tried different starting amounts, ranging from small amounts (about 200ng) to the highest amount the column accepts - 2µg. I don't know if smaller amounts can be amplified succesfully with that kit.
I have recently seen a poster and they performed a whole genome amplification of DNA after bisulfite treatment with the qiagen kit but I can't remember the amounts nor the author....
If you use smaller amounts of DNA, maybe the methyleasy Kit is even better, but I haven't tried so far (but it's at leats what Nick says....)
K.