No PCR products? - primer annealing time
I am a student looking for 2-gene by PCR-products plant AGL20 Piper sarmentosum Roxb (betel). I am using the primers AGL20, 5'-CCC-CAT-GTG-ATG-AGG-GGC-AAA-ACT-C-3 ', 5'-CCC GGA-TCC-TCA-CTT-TGA-TCT-AGA -- ACA-a ...
Problem, however, not able to produce all of the bands of PCR product, even after more than 20 countries) (all PCR products. DNA samples for PCR were used, extracted with the modified CTAB method and show very well in the electrophoresis gel. I have tried variations with the PCR mixture, including the increase or decrease of staff / MgCl / dNTP / Taq / primer concentration, but without result. Hot starts, so that the denaturation and annealing time were also considered. Annealing temperatures between 55-65 degrees i useC. Rees
I know that the plant is to produce PCR products becos older than me already, even though the products are degraded capability.
So what are the problems that be relevant R & D's solutions can?
Primer Annealing Time No PCR Products?
3 comments:
You are using the oligonucleotides of high GC content and the first of these documents a wide range of what is likely to prevent hybridization has. Check it out ... GG-cccc ---- q qq Gq was at this first. They know that binds G to C at a hairpin bend. I think the first method is your main concern, really. Redesign that I was not the band. Even with the GC content, you can really try FUND annealing temperature to about 70
Good luck, but I do not really need to redesign the first book.
Technology;
1. To change all of your advice to use or configure the response.
2. Try using a different PCR machine
3. You say you've changed the annealing temperature. If your PCR product (more than 2K) high, you may need to increase the extension of the time, I would leave one minutes per KB, approx.
4. can try to re-maps or other ingredients (NTP). Primers can with DNase or NTP could be contaminated .. End then try to reagenst a nearby lab or something lend.
5. Make sure that the extraction protocol did not work. maybe get a pair of primers and reinforce other region with the same genomic DNA
Theory:
the sequence of the primers, and perhaps develop new ones. This packet has to be wrong or something.
Although unlikely, some sequences of NCBI in May incorrect. also worth trying a new primer pair ..
that's all I can think of ..
Good luck
I do not know the model sequence, we do not know if all the model hybrizes track or not.
The first thing that comes to mind is the use of annealing temperature 50 ° C (the despair is my personal limit). If this fails, then you have a problem with:
1. their caps (the poor, confused and gave you is not the first right (s), providers rarely damage oligos) were freeze-dried or can be resolved not again to check your course properly.Of design and consulting.
2. Polymerase is wrong.
3. Staff is preparing a pollution that kills and order of reaction is something else. (You may need a stem with a little different than in the sequence orand the base could be a mutation or simply a different codon for the same amino acid).
Use a new proofreading polymerase. In this way, if you have a gap in the vicinity of the primer 3 'polymerase, or can cut a little late and expand the model.
Use a different response as a positive control (template and primers that have worked), so you can check the polymerase / dNTP
Use a second positive test: the model should have the more of a different set of primers Wotka has on them. This gives you the quality control. If you are a part of their workforce clean if it wants to be sure that no pollutants by ethanol precipitation or using an appropriate kit.
You can make nested PCR. With capsa DNA fragment that is slightly larger (and not merely reinforce a few cycles) and then use the "normal" primer to the desired fragment strengthened.
If you are a difficult problem and / or long model with a mixture of Taq polymerase Pwo + B.
Post a Comment