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To determine the exact TSS, you need to do primer extension assay, or do a 5' RACE to determine the 5' far end of the exon which can then be regarded as the TSS.
To predict whether the 1000 bp sequence derived from genome walking, you can use programs to predict promoters. There are many such programs, the one I use most is the Neural Network Promoter Prediction at http://www.fruitfly..../promoter.html.
#145273 How to find a promoter and TSS?
Posted
on 14 November 2012 - 07:06 PM
#107941 How is bisulfite converted DNA unstable over time?
Posted
on 25 April 2011 - 06:55 PM
It should be stable for at least 6 months if properly stored at -20C.
#139406 Bisulfite conversion conundrum
Posted
on 13 August 2012 - 09:50 PM
Your PCR conditions appear OK to me. The only thing the concerns me is the 60C Ta which is a bit too high for BSP.
If you use the same primers left from your colleague, there is also a concern of primer degradation.
A possible explanation for successful amplification of M.SssI treated DNA only is that the treatment step somehow causes DNA denaturing or fragmentation to some extent, which make subsequent bisulfite conversion easier because sodium bisulfite works on single stranded DNA only. That is why the initial denaturing step is critical and some early protocols even have to shear DNA or digest DNA by restriction enzymes.
If you use the same primers left from your colleague, there is also a concern of primer degradation.
A possible explanation for successful amplification of M.SssI treated DNA only is that the treatment step somehow causes DNA denaturing or fragmentation to some extent, which make subsequent bisulfite conversion easier because sodium bisulfite works on single stranded DNA only. That is why the initial denaturing step is critical and some early protocols even have to shear DNA or digest DNA by restriction enzymes.
#139301 Bisulfite conversion conundrum
Posted
on 11 August 2012 - 11:37 AM
Hi texasepigenetics, welcome to bioforum and to the methylation field!
First of all, please be aware that bisulfite PCR is much more difficult than regular PCR due to degraded DNA after modification and many constraints on primers.
Have you followed the same PCR protocol the other member used such as cycle number, Ta, etc? If yes, the problem may lie in your DNA. The Zymo kit should be reliable. A big problem with bisulfite conversion is not the issue of incomplete conversion but recovery of DNA.
Can you post your pcr conditions here? Often you need two rounds of PCR to see bands.
First of all, please be aware that bisulfite PCR is much more difficult than regular PCR due to degraded DNA after modification and many constraints on primers.
Have you followed the same PCR protocol the other member used such as cycle number, Ta, etc? If yes, the problem may lie in your DNA. The Zymo kit should be reliable. A big problem with bisulfite conversion is not the issue of incomplete conversion but recovery of DNA.
Can you post your pcr conditions here? Often you need two rounds of PCR to see bands.
#139586 Different primer optimalization for nested vs direct MSP?
Posted
on 16 August 2012 - 10:36 PM
For nested MSP, I think it is more appropriate to use universal outer primer set to first amplify bisulfite modified DNA regardless of methylation status. Then the same PCR product can be used as template for nested PCR using either the M and U primers. Nested MSP using the same M or U set will likely introduce huge bias upon two rounds of amplification if they have different amplification efficiency.
By inherent defect, I mean bisulfite PCR primers are not and sometimes cannot be optimized by the criteria of normal PCR.
Regarding cycle number, similar to RT-PCR, you depend on the intensity of resulted bands to judge the degree of methylation, or expression as for RT-PCR, a high cycle number tends to give you false positive results.
By inherent defect, I mean bisulfite PCR primers are not and sometimes cannot be optimized by the criteria of normal PCR.
Regarding cycle number, similar to RT-PCR, you depend on the intensity of resulted bands to judge the degree of methylation, or expression as for RT-PCR, a high cycle number tends to give you false positive results.
