Need Methylation MSP protocol - (Aug/28/2013 )

Hello, 

Just I would like to ask you some question regarding to my methylation experiment. I am new to this so, some of the question are stupid:

1- I have treated cells  with 5 aza and made RNA and would like to make DNA. We have Wizard (promega) kit. Is it good to purify treated DNA or is there other kit can be use?

2- if I purify the DNA. Do I treat all same , or I split to two one treated M and other U untreated?

3- What is the best website for MSP primer design? and predict CpG island?  

4- If you have a good protocol for Methylation MSP  could you please let me know?

-seeking_2012-

>>1- I have treated cells  with 5 aza and made RNA and would like to make DNA. We have Wizard (promega) kit. Is it good to purify treated DNA or is there other kit can be use?

The wizard kit is not very good for recovering bisulfite treated DNA. If you use some kind of kit for the treatment, the kit should include columns for DNA cleanup.

 

>>2- if I purify the DNA. Do I treat all same , or I split to two one treated M and other U untreated?

All DNA should be treated the same. If you need untreated DNA as controls, just one DNA sample is enough.

 

>>3- What is the best website for MSP primer design? and predict CpG island?  

There are several online tools for such purposes such as methprimer. 

 

>>4- If you have a good protocol for Methylation MSP  could you please let me know?

Try this one http://www.protocol-online.org/cgi-bin/prot/view_cache.cgi?ID=1088

-postdoc-

-seeking_2012-

-seeking_2012-