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modifications in bisulfite treatment


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#1 newinmsp

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Posted 28 February 2009 - 08:05 AM

Hi,
I am trying to use the protocol submited by labtechie in 2005, the one wich uses agarose beads.How do you do the washes??? Do you use the P1000? For me it is very dificult pick up all the solution (oil inclusive). I would appreciate any advice. Thank you

#2 MoB

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Posted 28 February 2009 - 11:15 AM

Hi,
I am trying to use the protocol submited by labtechie in 2005, the one wich uses agarose beads.How do you do the washes??? Do you use the P1000? For me it is very dificult pick up all the solution (oil inclusive). I would appreciate any advice. Thank you


Please don't get me wrong, but the agarose bisulfite protocol seems to be a very archaic method to produce bisulfite-converted DNA. Alkaline denaturation, mineral oil-layer, several washing-steps, high loss of DNA during the treatment and washing steps (especially for the smaller fragments), inhibition of downstream-applications by the agarose, melting of the beads before PCR...

My advice: Forget about it and try either the Qiagen EpiTect Bisulfite Kit or the Zymo Research EZ Methylation Gold Kit. Or if you want to save money, have a look into this paper: Reimo Tetzner, Dimo Dietrich, and Juergen Distler: Control of carry-over contamination for PCR-based DNA methylation quantification using bisulfite treated DNA, Nucleic Acids Res., January 2007; 35: e4. This seems to be also a very interesting protocol...

Hope that helps...

MoB

#3 newinmsp

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Posted 28 February 2009 - 01:47 PM

Thank you Mob,
The problem is that i was trying after the treatment to purify the DNA with the promega colums and I lose a lot DNA and then when i do the MSP when i have the bands they are so weak, so i was trying using differents amounts of DNA but nothing changes.... Now i am so confuss

#4 MoB

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Posted 01 March 2009 - 01:10 AM

Thank you Mob,
The problem is that i was trying after the treatment to purify the DNA with the promega colums and I lose a lot DNA and then when i do the MSP when i have the bands they are so weak, so i was trying using differents amounts of DNA but nothing changes.... Now i am so confuss


What exactly have you done with the Promega columns??? Have you used a kit for extracting PCR products from agarose gels?

Purification after bisulfite-treatment is done by washing the agarose-beads in a tube. It's quite a while that I have done the last agarose-beads treatment. I purified the beads in a 2.0 ml reaction tube, added 1 ml 1x TE, 0.2 M NaOH, or whatever buffer/solution is needed and incubate the tubes at RT for 10 minutes while shaking on a thermoshaker at 500 - 1000 rpm. After that the buffers were removed by pipetting them off the tubes. Take care that the washing- or desulfonation buffers are completely removed, if necessary spin them briefly and remove residuals with a smaller pipet. Residual sulfite and NaOH are strong PCR-inhibitors!

#5 newinmsp

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Posted 01 March 2009 - 05:31 AM

Thank you Mob,
The problem is that i was trying after the treatment to purify the DNA with the promega colums and I lose a lot DNA and then when i do the MSP when i have the bands they are so weak, so i was trying using differents amounts of DNA but nothing changes.... Now i am so confuss


What exactly have you done with the Promega columns??? Have you used a kit for extracting PCR products from agarose gels?

Purification after bisulfite-treatment is done by washing the agarose-beads in a tube. It's quite a while that I have done the last agarose-beads treatment. I purified the beads in a 2.0 ml reaction tube, added 1 ml 1x TE, 0.2 M NaOH, or whatever buffer/solution is needed and incubate the tubes at RT for 10 minutes while shaking on a thermoshaker at 500 - 1000 rpm. After that the buffers were removed by pipetting them off the tubes. Take care that the washing- or desulfonation buffers are completely removed, if necessary spin them briefly and remove residuals with a smaller pipet. Residual sulfite and NaOH are strong PCR-inhibitors!

Hi Mob!!!!
No, NO. NO, !!!!! i USED THE PROMEGA COLUMS when i did the treatment in eppendorf, nothing of agarose beads here!. As i was not having good results , because the bands were too weak , i decided to use the protocol with agarose beads....
My problem is that using the promega colums i lost a lot of DNA, i started the treatment with 2 ug of DNA and after the treatment i recover about 30- 60 ng. I used more vol. of DNA in PCR , but improved a little, so started thinking that the problem was in the bissulfite treatment. Pease I need help!!!!! thank you

#6 MoB

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Posted 01 March 2009 - 07:27 AM

Did you use the Promega Wizard Kit? I tested this kit several years ago and also got very low DNA recovery. Try a commercial bisulfte kit as recommended in an earlier posting. Conversion and purity are excellent and you won't have any troubles with low yields!

The problem lies not in the bisulfite treatment but within the subsequential purification. Just keep in mind that the DNA after bisulfite treatment is single-stranded, less comnplex and less flexible. If you use a DNA purification kit like the Promega Wizard Kit which is optimized for dsDNA the yields must be low. This is also reported in several publications. Try ultrafiltration (the linked paper) or one of the bisulfite-kits...

#7 newinmsp

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Posted 01 March 2009 - 03:26 PM

Did you use the Promega Wizard Kit? I tested this kit several years ago and also got very low DNA recovery. Try a commercial bisulfte kit as recommended in an earlier posting. Conversion and purity are excellent and you won't have any troubles with low yields!

The problem lies not in the bisulfite treatment but within the subsequential purification. Just keep in mind that the DNA after bisulfite treatment is single-stranded, less comnplex and less flexible. If you use a DNA purification kit like the Promega Wizard Kit which is optimized for dsDNA the yields must be low. This is also reported in several publications. Try ultrafiltration (the linked paper) or one of the bisulfite-kits...

Thak you very much, MoB!!!
I will try to convince my boss to buy one of this kits!!!
Iam using a conventional Taq polimerase from ultrachem, this could be a problem???
Other thing, I have to desing primers ( M and U) for USF1 and USF2 ( transcription factos) , how can i find the promoter region and then design the primers? Could be step by step, please




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