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Bisulphite trouble


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#1 epicrazy

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Posted 13 April 2010 - 09:23 AM

I am a first year grad, and did bisulphite sequencing for the first time ( 4 hr incubation @ 55C). This worked (I tested that DNA using control primers). I did it again for 48 samples! (and it didnt work, except this time I did 16hr@ 50C). I did it again for a third time on a few samples (4 hr incubation @ 50C) thinking my DNA was degraded too much, and it hasnt worked again ;) I am not sure what s going wrong. I have set up couple more tubes today, but I have covered my tubes with Al foil which I didnt do before. My DNA is ok ( i checked them on a gel before using it)
Briefly my protocol is:
- 2ug of DNA in 32.5ul of water + 1.1ul of 10N NaOH (freshly prepared) - incubate @ 55C for 15 mins
- Add 1ul of glycogen; 1.5ul of hydroquinone + 200ul of bisulphite (freshly prepared; ph is 5)
- Incubate at 50C for 16hrs or 55 for 4hrs
- Clean up using qiagen kit
- Desulphote
- neutralise

Any suggestions would be welcome!

#2 methylnick

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Posted 14 April 2010 - 04:03 AM

how are you assessing conversion has worked there?

#3 epicrazy

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Posted 14 April 2010 - 04:21 AM

I use a set of bisulphite primers that is routinely used in the lab. I was thinking I should test the bisulphite samples with normal genomic DNA primers to see if there are unconverted sequences (any DNA at all) and to make sure there is no PCR inhibiting substance in the prep?




how are you assessing conversion has worked there?






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