chip seq protocol - (Apr/26/2012 )

Dear all,

I would like to do Chip seq technique to my gene of interest. The problem I find many protocols about that and they use different lysis buffer like ( SDS lysis buffer,, or whole cell lysis buffer (abcam) ,, farnham cell lysis buffer)

In my experiment I will crosslink cells and carry on the Chip:

My question:

1- Does anyone have a protocol of chip seq with cancer cells like ( SW480, NT2)?


2- I am confused between using farnham lysis buffer or SDS lysis buffer whats the different ?


3- if I would like to work in only nuclei protien what lysis buffer and technique i shoud use?

many thanks
seeking_2012

I suggest you that do not waste your time with chip-seq.

Chep-exo is 30% better than that.
http://www.peconicge...20services.html

This company which is the owner of patent of chip-exo can do chip-exo for you with just 1400$.
this is the instruction that I have received from this company:

https://docs.google.com/open?id=1mrCDKYw2utJULE-mgrl3ATrALEkOXzjRCYYbOQ4Yppe4fwHbgaXhaI6Q40ML
https://docs.google.com/open?id=1mfUYRsc1qQvGNQYq1gDf_dUSd3VaD0fAcuiys8AONISC4vXr9YKkBMEkb5Kd

Babak from McGill

If you want yet to waste your time ok this is a sample I have:

https://docs.google.com/open?id=0B15muZJPLjJgbm81UG11X2pUbGlQb3llU2lzampldw

Hi memari,

Thank you for your replay.. and it is a good idea to use Chip-exo .. but for my project I have to stick with chip seq ..
I do not know if anyone can answer my questions above cause I need to know whats is the different:

2- I am confused between using farnham lysis buffer or SDS lysis buffer whats the different ?


3- if I would like to work in only nuclei protien what lysis buffer and technique i shoud use?

Farnham lysis buffer uses NP40 as a detergent to lyse the cell membrane but it not the nuclear membrane. The nuclei are then subsequently transferred to an SDS nuclear lysis/sonication buffer to lyse the nuclei and release the chromatin.

If you use an SDS lysis buffer from the start you can perform all the lysis and sonication in the same buffer, but you may get interference or background from cytoplasmic proteins.

Here's some good information:
http://www.abcam.com/index.html?pageconfig=resource&rid=11385
http://www.protocol-online.org/biology-forums/posts/33794.html

Hello there,

I have been doing ChiP assay using a kit from Upstate, millipore.
I completed the entire procedure and did PCR for GAPDH promoter sequence with positive control (RNA Pol II antibody) and negative control (Mouse IgG).
Surprisingly i got a band in both positive and negative . The band was fainter in negative, but then there was a distinct band after PCR.

Any help on what i should do right???

ivap