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positive control for MSP


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#1 epigenetics

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Posted 17 February 2009 - 12:34 PM

I am trying to generate positive control for my MSP expt. by treating DNA with sss1 (From NE BIOLAB) followed by bisulfite modification. protocol i am following which is give be NE biolab. But in my hand i could not make it 90% or more. I am finding bands in both M and U lanes.
How to make it exaustive, Anybody has experience in it?
thanks

#2 newinmsp

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Posted 17 February 2009 - 02:44 PM

Hi epigenetics,
I was trying to do the same thing using that enzime from NEbiolabs, but when i ran the PCR if there is any band it was too clear
I am buying a methylated DNA from millipore cat. number S7821. I will give you notices when it arrives...
Do you know how to get the primers using the meth primer program???? iam not sure if i am using it properly, because the primers i got have the same numbers of bp . How will i distinguish the M from the U in the polyacrilamide gel?
Any advice it is welcome!!!!

#3 MoB

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Posted 17 February 2009 - 11:45 PM

I am trying to generate positive control for my MSP expt. by treating DNA with sss1 (From NE BIOLAB) followed by bisulfite modification. protocol i am following which is give be NE biolab. But in my hand i could not make it 90% or more. I am finding bands in both M and U lanes.
How to make it exaustive, Anybody has experience in it?
thanks


I recently also had problems receiving a complete methylation using the NEB M.SssI kit. I determined a significant amount of unmethylated DNA by MSP for nearly all preparations.

There was an interesting topic posted just right before the server-crash. A member mentioned that they always obtained a residual of non-methylated DNA after M.SssI treatment. The problem seems to be the NEBuffer2 which contains a significant amount of MgCl2. In the presence of Mg2+ the methylation by M.SssI becomes distributive rather than processive. So they prepared their own reaction buffer without MgCl2. We have tested this protocol and got excellent results. Unfortunately there seems to be more problems with the NEB kit. But there is a new distributor: Zymo Research offers a M.SssI kit (E2010, 200 units) providing an optimized buffer (as they said) and interestingly recommending deviant reaction temperatures and incubation times. Kit was ordered, I keep you updated...

Hope that helps...

MoB

#4 epigenetics

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Posted 18 February 2009 - 07:14 AM

Thanks MOB,

Is it possible to get the protocol with the reaction buffer without MgCl2?
And if you get good result with Zymo kit, please let me know.
Thanks,

I am trying to generate positive control for my MSP expt. by treating DNA with sss1 (From NE BIOLAB) followed by bisulfite modification. protocol i am following which is give be NE biolab. But in my hand i could not make it 90% or more. I am finding bands in both M and U lanes.
How to make it exaustive, Anybody has experience in it?
thanks


I recently also had problems receiving a complete methylation using the NEB M.SssI kit. I determined a significant amount of unmethylated DNA by MSP for nearly all preparations.

There was an interesting topic posted just right before the server-crash. A member mentioned that they always obtained a residual of non-methylated DNA after M.SssI treatment. The problem seems to be the NEBuffer2 which contains a significant amount of MgCl2. In the presence of Mg2+ the methylation by M.SssI becomes distributive rather than processive. So they prepared their own reaction buffer without MgCl2. We have tested this protocol and got excellent results. Unfortunately there seems to be more problems with the NEB kit. But there is a new distributor: Zymo Research offers a M.SssI kit (E2010, 200 units) providing an optimized buffer (as they said) and interestingly recommending deviant reaction temperatures and incubation times. Kit was ordered, I keep you updated...

Hope that helps...

MoB



#5 epigenetics

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Posted 18 February 2009 - 07:20 AM

I used Meth primer to generate primers for MSP for 4 of my genes. But I got expected result only in One till now. Put your sequence (<5000 bp), then just check the boxes whether for BSP/MSP, Whether to use CpG island to generate primers, then Go. It will give you primer report. Sometimes it cannot predict primers. In that case i tried Methyl Primer Express from ABI.

Hope this helps.

Hi epigenetics,
I was trying to do the same thing using that enzime from NEbiolabs, but when i ran the PCR if there is any band it was too clear
I am buying a methylated DNA from millipore cat. number S7821. I will give you notices when it arrives...
Do you know how to get the primers using the meth primer program???? iam not sure if i am using it properly, because the primers i got have the same numbers of bp . How will i distinguish the M from the U in the polyacrilamide gel?
Any advice it is welcome!!!!



#6 epigenetics

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Posted 18 February 2009 - 07:21 AM

Ohh, and it uses primer 3 to determine bp of primers.


Hi epigenetics,
I was trying to do the same thing using that enzime from NEbiolabs, but when i ran the PCR if there is any band it was too clear
I am buying a methylated DNA from millipore cat. number S7821. I will give you notices when it arrives...
Do you know how to get the primers using the meth primer program???? iam not sure if i am using it properly, because the primers i got have the same numbers of bp . How will i distinguish the M from the U in the polyacrilamide gel?
Any advice it is welcome!!!!



#7 MoB

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Posted 18 February 2009 - 08:08 AM

Thanks MOB,

Is it possible to get the protocol with the reaction buffer without MgCl2?
And if you get good result with Zymo kit, please let me know.


Easy! Composition and concentrations can be found on the NEB website for a 1X NEBuffer 2 under 'Reaction & Storage Conditions'. Omit the MgCl2 for preparation of a 10X buffer, that's all...

BTW: a first test of the Zymo kit gave excellent results! No bands for unmethylated but strong bands for methylated after MSP!

Best...

MoB




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