Iím new in the field of methylation-specific PCR (MS-PCR).
Iím using MS-PCR to investigate methylation at imprinted loci.
I use EZ DNA methylation-Gold Kit (Zymo Research) to bisulfite convert my DNA (a 2.5 hour reaction as recommended by the company). I have obtained the primer sequences from another laboratory and they appear to work well in my hands as well. However Iím wondering how reproducible my MS-PCR results are.
Iím interested in calculating the unmethylated/methylated ratio of my DNA samples. How big differences in this ratio should I expect/tolerate if in run 2 identical PCR reactions with DNA from the same sample (and from the same bisulfate reaction)?
Some of my results are quite reproducible but sometimes I see differences in the ratio of 0.8-1. Is that way too much? And if so, what can be the explanation?
I'm using Puregene DNA purification Kit to isolate genomic DNA from cultured fibroblast cells. I have both tried the protocol for purification from 1-2 mill. cells and from 10-20 mill. cells. However my yield of DNA is really low.
In my first try I got between 20 to 50 ug DNA which I guess is okay (expected yield according to the protocol is 50-100 ug).
However, when repeating the purification on the same type of cells the yield was only 3-10 ug.
During the first try I did see the DNA precipitating and a small pellet was formed, however it wasn't that big a pellet as I would have expected from the number of cells.
During the second try I could hardly see any DNA precipitation or DNA pellet.
What can be the big difference between my two purifications ?
The cells have been treated similarly (left at room temp. in DNA hydration solution for ~2 years. Which is okay according to the protocol. Can this be a problem ??).
The only difference between the two trials that I can think of is the fact that I performed the RNase treatment and protein precipitation in a 1.5 ml eppendorf tube and a 4.5 ml tube respectively in the two trials. Should that make any difference ?
Both trials were performed using the protocol for purification of 10-20 million cells.
When I used the protocol for 1-2 million cells the yield was just as low, ranging from 1 to 27 ug.
I really hope that you can give me some advise to what have went wrong :-)