Multiplexing first round PCR in BSP - Any experience? (Feb/14/2007 )
Hi everybody out there in methylation-land,
I have thought about multiplexing four to six genes in my first round PCR for BSP, as I have only small amounts of template (and running out of taq way before a new order can be placed ). The Tms and the protocols are the same for all genes of interest in the first round.
Has anybody tried that? Does it work? I have done multiplexed first round PCR for MSP and it worked, but you'll never know...
Thanks for input,
if the regions you are looking at are distant from each other and there is no chance of cross amplification I can't see why not.
if your amplicon sizes are unique it would help the process along nicely.
I have not yet gotten to sequencing them, but on the gel the second round products look quite normal! At the moment I am trying to amplify two different fragments in round two in one well, one fragment is about 460 the other only 219bp long. Hope I can seperate them on the gel and purify them seperately. If your interested in the outcome, I'll post it here.
Please do krumel,
sounds like you are onto a biotechniques paper!
We'll see about biotechniques ... but if so, how do you want to be mentioned in the Acknowledgements section? With your screenname?
The gel is running...
Okay, I have still not gotten to sequencing the products, but the gel looks encouraging (see attachment).
I used all four primer pairs in one reaction-mix for the first round and then went on with two primer pairs per well in the second round. The image of the gel is not so nice (our gel-Dok is horrible), but the bands were really clear-cut when viewed by eye.
This reduces the amount of PCR reactions needed from 8 to 3 (for four fragments)! My boss will love me...
Wow, this sounds really cool - well done Krümel !
I'm really curious to see if this will work 100% - please post your progress.
Just looked at the gel - maybe increase the agarose content a little to obtain clearer bands?
Or is the Geldoc really THAT bad ? (if so you have my sympathy )
It is!!! It was broken last week - I had really hoped it couldn't be repared ... but somehow they did it
Only the good die young - all the evil seem to live forever...