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PCR not working - No amplification - (Aug/23/2010 )

I ran PCR a few times and I did not get any amplification. I know this because 1) I set up a gel electrophoresis and did not get a band and 2) I found that the concentration of my samples after PCR (in 30uL of water) ranges from 12-18ng/uL. Also, the samples have the same concentrations as my negative controls.

The PCR I set up was 25uL Phusion Mastermix, 2uL 10uM primers (forward and reverse primers were already mixed), 50ng DNA (.45uL), and 22.55 uL water(total volume = 50uL). The PCR program I used in a thermal cycler was:
10 minutes at 95C
15 cycles of 95C for 30sec, 54C for 30sec, and 72C for 30sec
5 minutes at 72C and then cooled to 4C

I then purified the samples using Qiaquick PCR Purification and I eluted in 30uL H2O. I then ran the samples on the gel, and I know the gel worked because the DNA ladder came out fine.

Do you have any explanations/suggestions for me?

-HSresearcher-

First, can you justify why you need 10 minutes at 95C? this will kill most of the activity of enzyme if i not wrong.
Second, why would you want to run just only a 15 cycle rather than normal 30 cycles?

I would say there are no amplification of your pcr product. The 12-18ng/uL is just your genomic dna + primers and perhaps some primer dimmer.

-adrian kohsf-

How were your primers designed? What are the Tm's of the primers?

I'm not so concerned about the 10 minute initial step; according to here, Phusion has a half-life of >6 hours at 96C. I agree that 15 cycles is cutting it a bit fine -- why so few?

-HomeBrew-

Hi,

I would also suggest increasing the number of cycles. Since PCR is exponential, the amplified product will increase and will be seen better on the gel after 25-30 cycles. It could be that your PCR is working but the product quantity is too little to be seen on the gel.

:)

-gt_ameya-

HomeBrew on Tue Aug 24 09:32:15 2010 said:


How were your primers designed? What are the Tm's of the primers?

I'm not so concerned about the 10 minute initial step; according to here, Phusion has a half-life of >6 hours at 96C. I agree that 15 cycles is cutting it a bit fine -- why so few?


:) ^_^ :P
Hi HomeBrew, thanks for pointed that out...

-adrian kohsf-

I have to say I agree with home-brew...I run a lot of qPCR and by 15 cycles I don't typically see any sort of product above background...

Also, what is the point of doing the qiaquick after the PCR? I don't think this is necessary for running out your product on the gel. If your amplicon is too small you also may lose it during this step as the kits say the DNA your purifying should be greater than 100bps long.

MM

-Mighty Mouse-