PCR failed no product.. help - (Mar/18/2010 )

I am using Qiagen Taq PCR master mix (concentration : 1x) however I did not get any product.. just wondering which is correct :

a)12.5ul of Taq PCR master mix + 9.5ul water + 2ul template DNA + 1ul primer
or
b)22ul of Taq PCR master mix + 2ul template DNA + 1ul primer

I am currently using option (a) but now I am wondering if I should add water to this since the master mix concentration is already at 1x

Anyone familiar with using this please advise..

Thanks

ocean_sky83 on Mar 18 2010, 01:02 PM said:

Normally i have heard of master mixes of 2X conc and above... but if u using option A, the conc of MM is only 0.5X in the final reaction which is not so great... may be then u shud try option B!!

Sorry my mistake, the concentration is 2x.. although the tube didn't indicate it but I printed out a material sheet from the website it has indicated 2x concentration..
So I guess option A is fine

ocean_sky83 on Mar 18 2010, 03:09 PM said:

Yes then the option A is correct...
If the reaction used to work earlier and not working now... then may be the primers have degraded.... but if its the first try.. may be there is something wrong with the primer design or the amplification cycles!!
Also the amount of template, conc of primer etc will matter....

What are you trying to amplify?

Also, am I right to assume you are only using one primer, and not a forward and reverse primer? If so, theres your problem...

ocean_sky83 on Mar 18 2010, 03:32 AM said:

wats the problem with using 2 primers?

No, what Bryan means is it is a problem to only use 1 primer.

From your original post, you state that you add only 1ul of primer- is that just one primer? Or two primers mixed together?

Yeah, sorry. In your post, to me it reads as if you only used one primer. The problem with this is you would not get noticable amplification of your product. PCR with one primer would be a linear reaction (not exponetial) and your maximum amount of amplified product would be ssDNA equal to your starting template. Probably not enough to show a band after gel electrophoresis. For standard PCR, you need to enhance both strands of your DNA, therefore needing a forward and reverse primer.

leelee on Mar 22 2010, 01:43 AM said:

If you only used 1 primer there's your problem. But if you did use both a forward and a reverse primer, sometimes (often) PCR needs to be optimized. Make sure your annealing temp is a couple of degrees lower than your Tm. Also, sometimes primer pairs just don't work and you need to redesign.

Yep. And if you have access to a thermocycler that will allow you to perform a gradient PCR, you can optimize your annealing temp that way.

microgirl on Mar 22 2010, 12:52 PM said: