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valeri_a

Member Since 23 Nov 2012
Offline Last Active Jun 09 2013 11:17 AM

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Chip pcr. are there inespecifics?

06 June 2013 - 07:41 AM

Hi guys!.
After IP and reverse cross linking and DNA purification, I done a standard PCR using control primers (GAPDH), wich I aspect  bands around  166 bp.

But.. This is my result of my standard PCR the samples are attached:

1- positive control (RNA pol)
2- negative control ( IgG)
3-  intesest target (HA)
4- Input
5- negative control of reaction  (water)

I don´t found explanation for this results. probably I have product in the imput, but I guess that I don´t have nothing what I looking for.  And  what about the amount of fragment of the IP product? is amazing. Is that because the IP is failed?, or it could be the pcr condictions??

another question , it could be that products forms dimeric structure??. or are those inespecific products?

thanks for your point of view!!!!

cheers,
Posted Image

How to check an antibody is ChIP grade?

17 May 2013 - 04:28 AM

how do I check that my antibody is ChIP grade?

Hi all,

I preforming a ChIP experiment, and to check the efficiency of my antibody, I will make a western blot after IP, but...... It is necesary to reverse the corss linking before?

Thanks!

name of a database to search homologous?

30 April 2013 - 05:09 AM

Hi all! does anybody know a database to search nucleotide homologous?
thank you! Posted Image

inespecific bands in nuclear fraction?

17 January 2013 - 03:58 AM

HI all,

I working with a cosntruct linked to GFP, I supposed that this protein could be a transcriptional factor. and for that, after transfection and adecuate treatment, I made a nuclear extraction to see my tagged protein in differents fraction by western blot. I saw very good expression in the cytoplasmic region, but also I saw strong band in the insoluble fraction, but near to 15 kDa. I tryied with antibodies against GFP and my protein, and appeared the same band.
I don´t understand, because GFP has a MW 25 Kda, and for that I supposed found bands higher than 25 Kda.
and because I don`t have too much experience in nuclear extraction, I don´t know if this signal could be an inespecific band, or histones, or anything else.

another thing. could I use lamin A/c as a marker of the soluble nuclear fraction??

THank you for your comments,

See u!

there is some way to replace MOPS in a buffer solution?

23 November 2012 - 12:37 PM

HI, I need hypotonic homogenization buffer, and I want to know if I can replace MOPS for any similar compound,

thank u !!

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