michele.xyz

Thanks for the heads up! I like!
I have just heard of another new generation GFP- antibody coupled to agarose beads which apparently is very good for CHIP (link below), and somebody else has recommended not to do CHIP with GFP antibody over night, as this increases the background.

http://www.chromotek...apr/gfp-trap-a/)

Another anti GFP I have come across in the literature is this one: 3E6 mouse monoclonal from Invitrogen, which comes as 100ug lyophilised.

I am a little bit unsure about what to make of the concentration of the rabbit antibody. I'll start off using 2mg resuspended in 500ul lysis buffer per tech rep, and I have no idea how much antibody to use so I'll have to establish that. If I get away using little, that would be great, but if I need 10ul/tech rep (which some people in our lab have used but for other antibodies), I need to think of something else because that's going to be so expensive!

Looking forward to your progress update!
Cheers

thanks- i am looking forward to your report

btw, how much antibody do you use for what concentration of cell lysate? do you CHIP in triplicate?

I used to run BAC gels which is a different type of acidic electophoresis, and all I did was I plugged the black lead into the red socket and the red one into the black socket and that worked just fine. I used a biorad apparatus too but I can't tell you what exact make they were, and they were big tanks for large gels.

I thought it was quite the fun to reverse the current.... Good luck!

Hi Trof,
once again your prompt advice is much appreciated. I think that's exactly what I'll do. Luckily, my primers have been designed and optimised by some company, so in theory they should work. Somebody else in the lab already used primers from them and they were just fine. The housekeeping genes are from the literature and have been successfully used (concentration etc optimised) in our lab by somebody else who did lots of RT-PCR (but he left unfortunately).
So in theory, there are established protocols and procedures in our lab which I'll follow exactly, but because I have never done it all by myself I am somewhat apprehensive.

But you are right, I think it is a good idea to first run the RT-PCR on some cDNA which was left by the other guy, it would give me an idea about what Ct of my target genes I can expect and give me some practice in handling. Also, I do have enough primers for 600 reactions, so they are not the limiting factor really. It didn't even occur to me.

btw- you are of course right, I would be diluting 1:9 to get a 10x dilution. 20ul cDNA in 180ul H2O. Just pure laziness.

  i'll drop a line once i have gotten somewhere with all of this.
thanks again.
m

Hi Trof,
thanks for your quick response.
You need to consider that there is usually no way to tell the abundance of your genes, so some amount may be OK for one gene, but insufficient for a low abundant.
That is exactly the reason why I was thinking of using all of the 5ug of RNA I have for the cDNA synthesis is because I am going to look at isoforms of transcription factors in response to different treatments. I suppose being transcription factors they won't be abundantly expressed so I thought by using all of my RNA I should hopefully get enough cDNA to see a difference- if there is any at all.
I don't have any experience at all with RT-PCR and expression levels of transcription factors and whether they can easily be detected by RT-PCR, so I thought the more I put in, the better chances to see something.
However, maybe 2.5ug would be already enough to detect the isoforms , and then I'd have spare in case everything goes wrong. But if the genes have very low expression levels and I have already used 2.5ug with no effect, then the other 2.5ug are kind of useless.

So how I would be doing the reaction is synthesise cDNA from either 2.5ug or 5ug RNA, using Superscript III RT with oligo dTs exactly as laid out in the protocol by Invitrogen, then dilute the cDNA 1:10 and run 5ul per well, prepare each condition in triplicate.

Do you have experience with RT-PCR for transcription factors? What would you do in my case?