Protocolpedia - The Encyclopedia Of Lab Protocols

hello viewers,

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 about it?

Kindly help me

Hope it will give some ideas. The bisulfite conversion degrades dna so that´s 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.

hello everyone,

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!