Bisulfite treatment of DNA follow by Sequencing - (Aug/05/2011 )

Hello Everyone,
I have found some region of interest(500bp-1000bp) by MeDIP-seq. Now, I want to confirm my MeDIP-seq result by Bisulfite treatment + PCR + Sequencing.
As far As I know, there are 4 critical steps in this experiment:(1)Bisulfit treatment of gDNA (2)Primer Design and synthesis (3)PCR amplification and purification (4)DNA sequencing (5)sequence alignment reporting. There are several silly questions in each steps that I need your help urgently.

1)Bisulfit treatment of gDNA: QIAGEN- EpiTect Bisulfite Kit will be used to bisulfit treatment of gDNA.
Question: how can I determine gDNA is real bisulfit converted?

2)Primer Design and synthesis:
Question: (1) Does ABI-Methy Promier Express Software v1.0 work well? Does there have any other software which is easy to use and work well?
(2) As my region of interest are two types:500bp and 1000bp segments, Some people said that 500bp segments should have 2 primers, but 1000bp segments should design 4 primers? Why? (maybe I misunderstanded what she said. )
3)PCR amplification and purification:
Question: The fellowing confition is ok?
PCR of bisulfite-converted gDNA:
AmpliTaq Gold® 10X buffer 1.0 μl
dNTP 2.5 mM each 0.8 μl
MgCl2 25 mM 0.8 μl
AmpliTaq Gold® (5 U/ul) 0.2 μl
Fwd and Rev Primer mix (2.5 μM each) 0.5 μl
Bisulfite-gDNA template (5-10ng/ul) 0.5 μl
Water 6.2 μl
Total 10.0 μl

PCR conditions for tailed primers:
95°C /5 min
5x (95°C /30 s; 60°C /2:00 min; 72°C/ 3:00 min)
30x (95°C /30 s; 65°C /1:00 min; 72°C/ 3:00 min)
60°C /85 min,
4°C Hold

(4)DNA sequencing:
Question:how much for 1 region of interest(500bp/1000bp)

(5)sequence alignment and analysis.
Question: How to do sequence alignment and analysis? which software should I use? Does there have any free software?
Thanks very much~~

qcshare

Question: how can I determine gDNA is real bisulfit converted?
Need to run a bisulphite PCR with primers that you know amplify converted DNA, your bisulphite kit should have this.

Question: (1) Does ABI-Methy Promier Express Software v1.0 work well? Does there have any other software which is easy to use and work well?
(2) As my region of interest are two types:500bp and 1000bp segments, Some people said that 500bp segments should have 2 primers, but 1000bp segments should design 4 primers? Why? (maybe I misunderstanded what she said. )

MethylPrimer express does a pretty good job of designing primers, the other way is manually by eye!


Question: The fellowing confition is ok?
pcr conditions look okay.

(4)DNA sequencing:
Question:how much for 1 region of interest(500bp/1000bp)

What do you mean>? are you cloning and then sequencing or direct sequencing? maybe direct sequencing judging by the tailed PCR step.

(5)sequence alignment and analysis.
Question: How to do sequence alignment and analysis? which software should I use? Does there have any free software?

google biqanalyzer

good luck

Thanks Nick,
There are some other questions:
1)It is necessary to design two pairs of primers when the region of interest is 1000bp?

2)I plan to use direct sequencing, As you said the price is judged by the tailed PCR step. Could you please give me something detail explaination about this? (I have never perform any PCR or related experiment).

3)Can I use Biqanalyzer to analyze direct sequencing data, I am little puzzled about "cloning and then sequencing" and "direct sequencing". What is the difference between them?

Thans again.

qcshare.

methylnick on Mon Aug 8 02:44:50 2011 said: