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michele.xyz

Member Since 30 Aug 2012
Offline Last Active Nov 28 2012 06:16 AM
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Topics I've Started

abcam 209 GFP antibody for CHIP- is there an alternative GFP antibody useful for

25 September 2012 - 06:01 AM

Hi all,
I am working on setting up CHIP using a GFP-tagged protein in C.elegans. I found that the ab209 Abcam rabbit polyclonal antibody is widely used for CHIP so I thought I'd give that a go. But: this antibody only comes in 50ul aliquot for about £200 and I have to optimise the CHIP before I can even start thinking about testing my genes. And I have already used half of it for Western Blotting with moderate success: I didn't get clean blots with this antibody. It is sensitive but not very specific I found on ordinary SDS-PAGE using the abcam protocol for blotting.

Hence my questions:
Does anybody have experience with another GFP antibody for CHIP-PCR? What are your recommendations?
Has anybody used this antibody for CHIP in C.elegans- what concentrations worked well?

I am using the protocol according to the Tissenbaum lab, A Mukhopadhyay - 2008

Any insight-expertise-suggestions would be greatly appreciated!

Thanks!

Upscaling cDNA synthesis using SuperScript™ III Reverse Transcriptase

30 August 2012 - 11:56 PM

Hi all,
I am planning to do RT-PCR on RNA samples (5ug) which are quite precious because they are not mine, they were a gift.
For the cDNA synthesis, I am going to use Superscript III RT with Oligo dTs. For the RT-PCR, we generally dilute the cDNA 1:10 and use 5ul per well, and set up each reaction in triplicates.
Since I am going to test 10 genes (including normalisation), and because the RNA is not mine and I can't go back and scrounge more of it, I calculated all the volumes I will be needing and it turns out the standard reaction volume of 20ul for cDNA synthesis with this RT is going to be enough for about 13 reactions. In case something goes wrong, I don't have a big margin to play around with the RT-PCRs.

So, I was wondering whether it is possible to upscale the reaction volume for the cDNA synthesis from the standard 20ul (in the protocol) to 30ul.
I haven't found any info anywhere- except there is another Invitrogen kit which allows for upscaling cDNA synthesis, but I'd rather not buy that if I can get around it.

Does anybody here have experience with upscaling the Invitrogen Superscript RT? Is it not recommended because e.g. the reaction is most efficient in 20ul? Or is it okay to increase the volumes?
I'm slightly hesitant to go ahead with it because it's not mentioned on any MDS or handbook on the Invitrogen website that it can be upscaled.

Some insight into this would be greatly appreciated.

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