Differences between MSP and BSP? - (Feb/28/2005 )

Thanks all for your help!

If I do enzyme digestion before bisulfite, do I need to purify the DNA after digestion?

I guess better clean up the DNA before modification. It's said pure DNA is important for complete conversion. check one of the pinned threads you will find the method how to do the clean-up.

That depends,

BSP is it's pro's and con's as does MSP. BSP can assay every CpG within your amplicon, MSP cannot.

MSP requires only two PCRs, one for methhylated and one for unmethylated and you get your result.

BSP only requires PCR and either direct sequencing or cloning and sequencing. These steps are more than just PCR. Direct sequencing is a little challenging and if it works it is usually the reverse primer that works. Cloning and sequencing is pretty straight forward but time consuming.

I have used methprimer for BSP primer design I have to say it was rather lousy and I pick by eye these days. I am not too sure if the authors have changed the parameters for BSP primers.

Good luck!

Nick

Dear Nick,

Is a water bath sonicator good for shearing DNA in a CHIP assay??. I use "SUPER RK 103H from Schütt labortechnik, i sonicate DNA at 100 strenght for 2 mins + 2 mins in ice cold water (i place the samples on ice after the first 2 mins). The DNA smear on the gel looks the same with sonication time points of 1.5+1.5 mins or 2+2 mins or 3+3mins. Could you please explain.

Thank you.
sridhar.

I am not too sure what you mean by the DNA smear looks the same....what is the approximate size of the smear? I have not tried using a water bath sonicator but I have heard it is better than the the conventional tip sonicator that we routinely use.

are you able to post a image to the board for all to see? From the info you have given, it could mean that the settings you used have completely sheared the DNA to completion or depending on the size, not sheared the chromatin at all.

Nick

Dear Nick,

The gel image is in the attachment, consider the tabular column on the left page and the gel images on the right page, ignore the rest. I used "1 Kb Plus DNA ladder" but the marker is not clearly visible. The smear in "lane 9" starts exactly at 100 and ends at 1000pb, this is shearing at 100 strenght for 1.5 mins with a break for 1 min on ice and again sonicating for 1.5 mins( i make sure that the water in the water bath sonicator is ice cold). However there is no big difference between all the lanes.


I used rat cortex cells from Embroynic day 18 for the sonication standardizing expts but i try the same parameters on mice cortex cells from Embryonic day 14, do think that the shearing efficiency is the same? please note that E14 has Euchromatin and E18 has heterochromatin.
Please reply back.

sridhar.

hi sridhar,

this confirms my understanding that water bath sonicators are more efficient than the tip sonicators.

You want to see a smear ranging from 100-1000bp after sonication. I can't see your ladder however, it seems you have fully sonicated your chromatin with the shortest condition, there are some high molecular weight stuff but as you can see the majority of the smear is within the right size range, have these been RNAse treated? it seems you have stackloads of DNA but this could be because you are also seeing RNA.

it looks fine, I think I will ask my PI to get a waterbath sonicator!

Nick

hi nick,

the samples in my gel image are RNAse treated. I have bands in the PCR reaction for the"no Ab" control of my CHIP, however these bands have a low intensity when compared to the +ve controls. Will the tratment of "agarose beads" with BSA completely remove the bands in my PCR reaction?? can you commet about that??

Is there an alternative method to purify DNA other than Phenol: chloroform extraction before proceeding for a PCR in a CHIP assay?

sridhar.

Dear nick,

I have a important question to ask you, my sonicated products were run on a 0.8% gel at a very low voltage for about 1.5 hrs which gives me a thick smear between 200bp to 1.2kb, when i run the same samples at a very high voltage, i have a very thin smear all throughout the gel, does it mean that my samples are properly sheared or not, please reply this back.

sridhar.