Serious problem with bisulfite modification - (Nov/17/2007 )

Hi everyone!

I'm a new user!
I've a ver big trouble with bisulfite treatment.
I have 2 CpG islands to investigate in my gene.

I've tried nine treatment:

1-2 like the kit chemicon said (T° 55°C for 12 h)
3- kit chemicon with some modification (t 55°C for 6 h in agarose beads)
4- kit chemicon with some modification (pre boiled 100°C, and incubation with bisulfite at 55°C for 6 h)
5- kit chemicon with some modification (T° 95° for 1 h)
6-7 like the kit Sigma said (one T° 65° for 90 min, one T° 65 for 180 min)
8- I've tried with Na-metabisulfite "by my hand" (T°65° for 5 h)
9- another one with kit chemicon (pre boiled 100°C, and incubation with bisulfite at 55°C for 6 h)


I've no modification!!!!!!

Only for the tratment 4 I had some modification but not very significant.

I've tried 3 different set of primers.
The second seem to work only for 1 island, I have a good amplification but after cloning and sequencing I have the same sequece without conversion.
The third set of primer amplify only the island 2, this primer have 3 T at the end for capture only the modification sequence, next week I'll try to cloning.

In a first moment I thought that was a problem of denaturation and so the bisulfite wasn't able to work, but after ther treatment at 95°C and a good PCR I think it's impossible.

Maybe is it a problem of primer design?
Where is my mistake?
I've tried every protocol....Please I'm desperate!!

Thanks a lot!

Cheers!

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I understand your pain.

your primers are the issue, where have you sourced them?

do not throw away your DNA, i think there would be material there for you to amplify from.

get good primers!!! and your woes will go away.

Nick

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Thank you Nick!
So you think that the problem is in the primer set, isnt' it?

Now I use 2 set:

1- Without C in the oligo's sequence to try to capture everything (modificated, unmodificated)
2- With 3 T at the end

My PCR cicles are:

I PCR:

94°: 2'
94°: 30''
42°: 30''
72°: 3'
72°: 7'

for 35 cicles

II PCR nested

94°: 2'
94°: 30''
42°: 30''
72°: 45''
72°: 7'

for 35 cicles

What do you thing? Maybe is the annealing temperature too low?

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42C seems awfully low,

how did you design your primers?

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