the mystery of the haunted pcr - Did my new preps degrade? Did my PCR fail? Or something more sinister? (Sep/09/2008 )

Hello,

My PI went on vacation for two weeks with instructions for me to take my DNA preps for 24 fly lines, amplify them with 10 different primers, and sequence the resulting data. Unfortunately I only got through two of the primers until I ran into a horrible problem.

Procedure:
I used the standard quick genomic fly prep, here: http://www.fruitfly.org/about/methods/inverse.pcr.html to make my preps, which are only 2 wks old. I followed this protocol exactly.
I then run a PCR with MgCl2, 10x buffer, dNTPs, Taq, millohm water, all the normal stuff... the primers are ones that he designed, forward and reverse...
I run the product on a 1% agarose gel at 5v/cm....
If the product produces nice bright bands, I take the remaining PCR products in the tube, clean them using the QiaQuick PCR Purification kit, and send a sample of them off for sequencing. I send the amplified products of the first four lines, with the forward primer and the reverse primer, for a total of eight sequences. If these sequences come back ok, I send the rest.

However, by the time I got to the second primer set, two of the bands on the gel were noticeably dim. Subsequent PCRs and gels showed that the two bands (next to each other) grew dimmer and dimmer... until they finally went out. And, to make things worse, the bands on EITHER SIDE of these two went dim and then stopped amplifying. It was like it was spreading out from those two.

I remade the preps that had stopped amplifying. I spec'ced the troublemaker preps and found that there was basically no DNA in them, even though they had PLENTY of DNA two weeks ago. I spec'ced the new preps and found that there was definitely some DNA in there. However, the new preps won't amplify either.
Before they stopped amplifying, I sent the first four lines of the third primer set to be sequenced, and it came back like the attachment. The first line is Fly Line 1 with Forward Primer, the second is Fly Line 1 with Reverse Primer, the third is Fly Line 2 with Forward Primer, the fourth is Fly Line 2 with Reverse Primer.

Sorry for this long-winded post, but I would welcome any insights you guys have. Just telling my PI "Oh, I suddenly couldn't amplify anything, and making new preps didn't change a thing!" won't really cut it.

Thank you so much!

Oh, and I should add that the ones that do still amplify do so very weakly, and have a lot of weird nonspecific bands. I tried running the PCR at a hotter temperature, as a gradient showed that these primers were most efficient at temperatures over 66º in the elongation stage. That didn't get rid of the bands.

Here's a picture of one of my gels, sorry it's so bad.

I am so desperate and will be so grateful for any help or advice.

I will be your internet friend. I will bake you internet-cookies. I will let you have my internet-parking-spot.

Drop me your primer sequence to my email box. I would like to have a look first.

typical problem: change your dNTPs. many times y dNTPs go bad and my PCR reactions stop working, change em and they start working again.
hope it helps!

I differ in opinion to mjolner. If there are problems to a PCR, it is often the template which is the primary cause, rather than any other component of the PCR mixture.

Do you have a control PCR, one that uses your template and you know will work? You need a positive PCR control. DNA that is not clean enough will not amplify either.

If you have aliquoted your reagents, I would throw every thing away and start with a fresh batch. New dNTP, new PCR buffer, new MgCl2....Things do get contaminated and degraded, it is natural. Debugging the problem is a hassle as you can imagine. The key to this kind of problems is the ability to start anew and just throw all the old reagents away. Thus the importance of aliquoting your reagents. The polymerase must be handle with extreme care.