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DNA yields after Bisulfite Treatment


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#1 sri2010

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Posted 28 October 2010 - 11:33 AM

Just treated my DNA with the BS Modification Kit from Sigma Aldrich.

I did a spec: the DNA concentration came to 6.310 ug/ml, Protien -9.439, DNA/Protien Ratio was .286.

The values I started with:

DNA Ratio: 1.714 DNA concentration: 50.28 ug/ul Protien: 3.553 ug/ul

I'm running a PCR tomorrow, and hopefully I will get bands.. but can anyone please share some insights on how to optimize this better?

It was suggested that run more cycles..what do you guys think?


Also, does http://www.urogene.o...mer/index1.html, I copied my 2.8 kb sequence, does this only design the primers around the CpG islands? I'm a little confused (even after reading the insert on how the program selects the primers) Because thats what I want..I want the primers to be designed around the CpG island. Not sure how the PCR will work out tomorrow...any advice would be greatly appreciated.

Thank you for your help in advance

#2 ElHo

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Posted 29 October 2010 - 01:13 AM

bisulfite conversion degrades dna so thatīs why a great amount of your input dna will be lost. Converted dna cannot be quantified by spectrophotometry as it is no longer double-stranded (complementarity is lost ddue to conversion). You often have to perform two rounds of pcr to get visible bands after agarose gel electrophoresis.

#3 sri2010

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Posted 29 October 2010 - 07:05 AM

bisulfite conversion degrades dna so thatīs why a great amount of your input dna will be lost. Converted dna cannot be quantified by spectrophotometry as it is no longer double-stranded (complementarity is lost ddue to conversion). You often have to perform two rounds of pcr to get visible bands after agarose gel electrophoresis.


oh.. okay, that makes sense. 2 rounds of PCR? = how many cycles?

#4 MoB

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Posted 01 November 2010 - 02:57 AM

bisulfite conversion degrades dna so thatīs why a great amount of your input dna will be lost. Converted dna cannot be quantified by spectrophotometry as it is no longer double-stranded (complementarity is lost ddue to conversion). You often have to perform two rounds of pcr to get visible bands after agarose gel electrophoresis.


I quantify my bisulfite-converted DNA always on a NanoDrop before running any PCRs and this works pretty good! Use factor '33' for ssDNA instead off '50' for dsDNA...

#5 fnad

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Posted 09 February 2011 - 03:35 PM

Hi MoB,

I'm new at this methylation-related stuff. When you use nanodrop in 33 (SS mode), what would be the number to look for in the 260/280 and 260/230? Is it also ~1.8 like DNA in double strand mode? I did measurement with factor 33, but it spits out bizarre number on those absorbance ratios which makes me think my conversion was bad. Hope you can share some thoughts. Thanks a lot!

FNAD

#6 MoB

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Posted 10 February 2011 - 04:26 AM

I'm new at this methylation-related stuff. When you use nanodrop in 33 (SS mode), what would be the number to look for in the 260/280 and 260/230? Is it also ~1.8 like DNA in double strand mode? I did measurement with factor 33, but it spits out bizarre number on those absorbance ratios which makes me think my conversion was bad. Hope you can share some thoughts. Thanks a lot!


I have no experiences with the 260/230 ratio. After bisulfite-treatment and purification all residuals from DNA extraction should be washed out, so this ratio is no longer interesting for me. The only thing I carefully look at is the 260/280 ratio. For all types of bisulfite-converted DNA I've found this ratio to be in between 1.95 and 2.2. If the ratio is smaller than 1.9 I would think you have a conversion problem. Quite interesting: for completely methylated DNAs the ratio is higher (close to 2.2), while for low methylated DNAs (for instance WGA-DNA or sperm-DNA), the ratio is more to be in the range of 2.0!

Hope that helps...

MoB

#7 fnad

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Posted 10 February 2011 - 07:36 AM

I have no experiences with the 260/230 ratio. After bisulfite-treatment and purification all residuals from DNA extraction should be washed out, so this ratio is no longer interesting for me. The only thing I carefully look at is the 260/280 ratio. For all types of bisulfite-converted DNA I've found this ratio to be in between 1.95 and 2.2. If the ratio is smaller than 1.9 I would think you have a conversion problem. Quite interesting: for completely methylated DNAs the ratio is higher (close to 2.2), while for low methylated DNAs (for instance WGA-DNA or sperm-DNA), the ratio is more to be in the range of 2.0!

Hope that helps...

MoB
[/quote]

Thanks a lot MoB!

I'm actually getting 260/280 around 3.0 - 4.0.. so would you think that's also an indication of bad conversion? Or maybe there's contamination there? I'm pretty sure I started out with good DNA, and I thought I did the bisulfite conversion OK - because I used the Epitect Bisulfite Kit which has a pretty straightforward protocol.. so I wonder where I went wrong there.. Maybe you can help me with more advise? Thanks!!

FNAD

#8 sri2010

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Posted 10 February 2011 - 08:31 AM

I just got my Bisulfite reaction to work after 4 long months yesterday, seemed the problem the whole time is the amounts of DNA you are putting into the BS reaction, try varying the amounts, I know the kit suggests the "optimum" amount, but that doesnt always seem to work. Hope this helps. I didn't use the Epitect kit, I used the Zymo Research kit.

Although I wouldnt use the spec value to determine whether the DNA conversion worked. Try using the primers (depending on which method you are doing MSP vs BSP) in the PCR and see whether it amplifies, that is really the only way you would know whether the conversion worked.

Good luck!

I'm actually getting 260/280 around 3.0 - 4.0.. so would you think that's also an indication of bad conversion? Or maybe there's contamination there? I'm pretty sure I started out with good DNA, and I thought I did the bisulfite conversion OK - because I used the Epitect Bisulfite Kit which has a pretty straightforward protocol.. so I wonder where I went wrong there.. Maybe you can help me with more advise? Thanks!!

FNAD
[/quote]

#9 fnad

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Posted 10 February 2011 - 11:48 AM

Thank you for the suggestion, I appreciate it!

fnad

[quote name='sri2010' timestamp='1297355512' post='100185']
I just got my Bisulfite reaction to work after 4 long months yesterday, seemed the problem the whole time is the amounts of DNA you are putting into the BS reaction, try varying the amounts, I know the kit suggests the "optimum" amount, but that doesnt always seem to work. Hope this helps. I didn't use the Epitect kit, I used the Zymo Research kit.

Although I wouldnt use the spec value to determine whether the DNA conversion worked. Try using the primers (depending on which method you are doing MSP vs BSP) in the PCR and see whether it amplifies, that is really the only way you would know whether the conversion worked.

Good luck!

#10 MoB

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Posted 15 February 2011 - 11:36 PM

I just got my Bisulfite reaction to work after 4 long months yesterday, seemed the problem the whole time is the amounts of DNA you are putting into the BS reaction, try varying the amounts, I know the kit suggests the "optimum" amount, but that doesnt always seem to work. Hope this helps. I didn't use the Epitect kit, I used the Zymo Research kit.

Although I wouldnt use the spec value to determine whether the DNA conversion worked. Try using the primers (depending on which method you are doing MSP vs BSP) in the PCR and see whether it amplifies, that is really the only way you would know whether the conversion worked.

Good luck!

I'm actually getting 260/280 around 3.0 - 4.0.. so would you think that's also an indication of bad conversion? Or maybe there's contamination there? I'm pretty sure I started out with good DNA, and I thought I did the bisulfite conversion OK - because I used the Epitect Bisulfite Kit which has a pretty straightforward protocol.. so I wonder where I went wrong there.. Maybe you can help me with more advise? Thanks!!

FNAD



From my experiences it's very difficult to produce a 'bad converted' DNA, especially with bisulfite-kits like the EpiTect- or the Zymo-kit. Either the DNA is converted or it remains completely unconverted. For a partially converted DNA I had to dilute the bisulfite reagent at least by the factor 1:10. And even then 260/280 ratios were in the range of 1.9 to 2.1. I have never obtained ratios around 3.0 to 4.0. Do you use carrier (RNA, glycogen, BSA, poly(d)A)? Maybe this might have an impact on the UV measurement?

#11 fnad

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Posted 16 February 2011 - 07:05 AM

Hi MoB,

I didn't use carrier RNA because I started with >100 ng DNA. I guess a way to find out at this point is to do PCR and see if it works or not..
Oh and by the way, when you nanodrop the converted DNA..did you get an accurate quantification? say, if you start your conversion with maybe 200 ng of DNA, did the nanodrop reading show that you end up with the same yield of around 200 ng too...

Thank you!!

FNAD

From my experiences it's very difficult to produce a 'bad converted' DNA, especially with bisulfite-kits like the EpiTect- or the Zymo-kit. Either the DNA is converted or it remains completely unconverted. For a partially converted DNA I had to dilute the bisulfite reagent at least by the factor 1:10. And even then 260/280 ratios were in the range of 1.9 to 2.1. I have never obtained ratios around 3.0 to 4.0. Do you use carrier (RNA, glycogen, BSA, poly(d)A)? Maybe this might have an impact on the UV measurement?
[/quote]

#12 Boba

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Posted 12 March 2011 - 08:45 PM

Try measuring your bisulfite converted DNA in a NaOH solution using a factor of 33 to calculate concentration.

#13 vilperte

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Posted 05 February 2013 - 04:40 AM

Hello!

I'm having the same problem! I couldn't amplify any of my 5 pairs of primers.

I started with 200ng and after the bisulfite conversion I ended up with 40ng. I used Nanodrop to quantify my samples and the 260/280 ratio ranges from 2,65 to 3,17.
I'll try to convert the DNA again to see if it helps me. I used RNA as carrier, so I'll do it without the RNA this time.

#14 Epigenome1

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Posted 07 April 2014 - 05:35 PM

I am following your discussion here. I have very similar problem. My question is how do you describe your gel?! Was there a smear or nothing at all?




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