33 replies to this topic

[Nephrite]

Dear all, this is my contribution to the topic: I have 4 primer pairs, amplifying 4 different products with length 259, 172, 117 and 97 bp from two different genes, i.e 2x2, so they are actually re-designed.
With all of them I have cross-dimers of 50 bp, in positive and in negative samples - I run a gel only with empty samples (no template) with both primers together, with F only, and R only. The band appears only when two primers are together. I do not have a problem with my housekeeping gene, which tells me that reagents are OK
The annealing temperature I use is 55 degs. The templates does not contain a lot of CG.
What I have done so far, but did not work:
1. Increase of annealing temp. to 60 deg. - dimers are there, but sometimes real product is missing.
2. Dilution of primers - same problem.
3. Titration of Mg++ - same problem.
4. Different combinations of these factors - same problem.

Tomorrow I decided to increase the template 10 times - and see what will happen.

1. Can anybody explain me what is the reason about this? When designed the primers I checked for secondary structures and cross-dimers - the program said they are OK. Then why???
2. How DMSO affects the reaction? I will try it tomorrow.
3. The two-step PCR that sillybiochemist wrote about - 1-15 rounds of PCR means cycles?

[dejay]

hi, i'm a beginner in molecular biology and am stuck with the interpretetion of PCR results.. I keep getting two bands(1500 and 750bp),i want to identify the bacteria, iused universal primers to get result but unwanted band of 750bp is there all times, i tried different anealing temp from 55-58 but result was same.  just have no idea why they are different size!!??),
many thanks for any help

[michael_mes]

Hi dejay!

You need a positive control, to test you PCR system. If primers and reaction conditions are good, then you should get only band of 1500bp.
Your 750 band may be due to contamination or presence other bacterial clone. One way or another, you must test your system (make primer blast, PCR in silico etc.).

[cancerlab]

You can use a hot start at the beginning and an extension at the end of your run. That will prevent primer dimers.