Primer Dimers

[Lisa]

Hi!

I am pretty new to molecular biology so please forgive any gaffes. I am looking at the chloroplast trnL intron, chloroplast rbcL-accD intergenic spacer and rDNA ITS (ITS1 +5.8S + ITS2) in a wild buckwheat.

The primers I am using are all universal plant primers and have been used in a closely related genus (as well a at least one species in my genus). The Tm of each primer is between 62 and 55 although they have been calculated at 55 to 52 on various websites. I am also using the Sigma Aldrich REDExtract-n-Amp Plant PCR kit. It is supposed to be idiot-proof. However, I am getting primer clouds (dimers?). Oddly enough, I was not getting them with my first kit. They started to appear with the second and, interestingly, they initially appeared as very faint bands that got brighter and brighter with each reaction.

I altered my primer concentrations and my template concentrations to no effect. I made a completely new set of primer working solutions. I ordered new primers from a different company. That didn't work. I really don't have the option to modify my reagents so that has not been done. However, I did play around with the thermocycler temps as well (see below).

The first set of temps were suggested based upon a similar study done with a closely related genus. The second method was programmed after the first method gave me no specific product (in fact, I got absolutely nothing). But then I learned I should have diluted my template. Nevertheless, before I realized that, the second method was used.

Someone then told me that the second method was way, way wrong because it did not incrementally decrease to 55C, but rather JUMPED to 55C after 20 cycles at 65. Thus, the third method was born. Remember when I said I was getting product with the first kit? Well, that was also using the second method of thermocyler temps. Does any of this make sense (?!?!) because I am losing it. I am now awaiting a third kit. If this kit does not help me strike gold, I will have to abandon this component of my thesis and I really, really wanted to do this.

Sorry for the novel. Does anyone have any advice?
Thanks a bunch!
Lisa

METHOD 1
Step 1 94 2 min
Step 2 94 45 sec
Step 3 55 1 min
Step 4 72 2 min
Repeat steps 2 – 4 29 times or, if yield is low, increase to 35
Step 5 72 6 min
Step 6 4 Hold

Method 2
Step 1 94 2 min
Step 2 94 45 sec
Step 3 65 1 min
Step 4 72 2 min
Repeat steps 2 – 4 20 times
Step 5 94 45 sec
Step 6 55 1 min
Step 7 72 2 min
Repeat steps 5 – 7 20 times
Step 8 72 6 min
Step 9 4 Hold

Method 3
Step 1
Step 2 94 45 sec
Step 3 65 1 min (in ½ degree increments every minute to 55)
Step 4 72 2 min
Repeat steps 2 – 4 20 times
Step 5 94 45 sec
Step 6 55 1 min
Step 7 72 2 min
Repeat steps 5 – 7 20 times
Step 8 72 6 min
Step 9 4 Hold

[Aga]

It is hard to help in this situation because it's hard to assess your results not actually seeing them.

First of all I would try to make it simple. When I have some unspecific products in my standard PCR I try to increase annealing temperature, but up to 62°C (I never got product using 65°C). For more stringent temperature regime I use usually 60°C (off course it depends on primers melting temp.).

What about your expected product size? Is it very long? There is usually little problem with amplification of short products, with longer products annealing time should be increased and Pfu polymerase is usually recommended instead of Taq.

If you have only primer-dimers in the first cycles of your reaction they will continue to amplify in next cycles - so the final amount of primer-dimers will increase.

Does the kit you're using contain hot start? If yes, you definitely have to increase initial denaturation time (to 10 min). I've just read that this kit contains hot start polymerase (I hope I checked it right). You won't activate polymerase if you don't use pre-incubation time (usually 10 min).

Based on what you wrote, not knowing product lenght, I would try to use annealing at 60°C during all the cycles - for start, and see what happens. And 10 min. initial polymerase activation.
If what I checked about the Sigma-Aldrich kit was right, you can add only initial pre-incubation and PCR should work ar 55°C. Check it out, you have nothing to loose besides several ul of reagents.

There is another issue - template DNA quality. Be sure that for assay development you always have good quality template - also in terms of purity. DNA frozen and thawed several times is not recommended.

[Lisa]

Hi Efraim,

I want to thank you for your response. Because I wasn't getting any replies, I sort of gave up hope and hadn't checked back until today. I appreciate your insights.

To answer some of your thoughts, my target site is roughly 600bp. The kit I am using (as described before) does not suggest a 10 min initial denaturation period in its protocol. Three minutes is the recommended period of time for that.

As for the Ta, I lowered mine significantly (51) based upon the Tm of a new set of primers I ordered and that seemed to improve things a bit but still not to the quality I was initially seeing.

I think you are right about the template. However, everyone I have spoken to says this kit is very robust. The extraction methods and solutions in it are supposed to give me a template that should keep for about 6 months. Nevertheless, the kit is really only as good as what goes into it, I suppose.

Anyway, I have since gone through a third kit and as this has gotten expensive for me, I have had to abandon this component of my project.

Thanks again.
Best