BS PCR..please help - DNA methylation analysis (Sep/01/2009 )
epimaster on Mar 31 2010, 11:42 AM said:
1. extracting DNA from cerebellum tissue by qiagen kit(during extraction I Used RNAse for avoiding any RNA)
2. reading UV, that shows me the concentration of THe DNA is great always.
3. treatment and clean up by the same kit, EZ DNA methylation gold kit, Zymo research
4. doing PCR with TaKARA prime star tag polymerase,
always I do normal PCR withe designing primers from methyl primer express saftware
PCR condition as TAKARA kit said:
38 cycles for
98 for 10 sec
55 for 5 sec
72 for 1 min
at the end gel
my result always is very faint smear for my interested samples but very nice band for control group, I mean un-bisulfite treated DNA
I'm soo appreciate if any body here can guide me , please.
Farimah
How much input DNA are you using per reaction and how much converted DNA are you getting? Are you denaturing with the zymo kit with PCR prior to working with the Takara polymerase? what type of carrier are you using?
Here are some preliminary suggestions:
1. Check for full conversion
2. Check for DNA impurities/improve purification
Hello again epigenius,
After extracting DNA by qiagen kit, I take 2μg of DNA as 20μl volum(az the Zymo kit has said) for treatment process and washing, and at the end I have 10μl of DNA(the last step of the kit) but I don't know if it is converted completely or not? how I can check it? and then I use 3μl of this (10μl) for PCR using TAKARA prime star,
and if you think how I have to purify my DNA after treatment (the kit said this DNA is ready for PCR), If I have to purify my DNA after each step? I mean after extracting and after treatment? If you suggest any purification way for purifying my DNA?
Thank you so much for your attention.
it is not necessary to repurify your samples after conversion, the kit does this for you.
One of the major problems is the primers you are using for PCR amplification are not properly designed, what primers are you using? are they specific for bisulphite converted DNA?
N
methylnick on Apr 14 2010, 05:10 AM said:
One of the major problems is the primers you are using for PCR amplification are not properly designed, what primers are you using? are they specific for bisulphite converted DNA?
N
Hey Nick, Thanks for reply
I used Prime star TAKARA for PCR, without any results, but I changed it to TFI, finally I got band but with a very regular polymerase, Tfi,, my advisor is worried about that because he belives this polymeraze has verry errors during polymerization!! and interesting part is that I got very nice bands with both of my primers specific and non-specific for bisulphate.
the error rate with standard Taq is unlikely to affect your amplicon of interest because they are short (less then 1kbp).
The proof reading taq's fall over because it is copying off a hybrid DNA containing U's that is why standard Taq works fine for bisulphite converted DNA.
Nick
Hello everybody, PLZ help me,
I was working on CpG methylation stuff, and from your guides, especially MethylNick, I finally could get very nice bands, my advisor and me became sooo happy and and I introduced your site to him too, we both thought that the most part of the work has been done, but... sad.gif .but I can not get any sequencing result from PCR product,,, I like to tell a little more about my procedure;
I extracted PCR products by gel extraction kit (qiagen) and directly I took 9μl of that and 3.2μl of forward primer in one 0.5 ml tube and send to sequence, but just untreated samples(without any bisulfite treatment) had results. I thought maybe there is one agent in my samples whihc does not let sequencing does well job, so I washed my samples(before DNA is treated with bisulfite with zymo kit, then amplified by PCR, and PCR product was extracted by kit) then I put 9μl of that in to a 0.5 ml tube and I add 3.2μl of reverse primer instead of forward primer and sent for sequencing again, the same result, I mean no any sequencing result for bisulfite treated samples.
So, PLZ let me know what you think? it is important that my PCR products are very short, <200bp so if you think I have to amplify my PCR products before sending to sequence?
I'm disrupted too much, my great friends, PLZ give me your ideas,
Thanks