I am currently working on primer optimization of 2 genes and I got these faint but specific bands. I haven’t been able to get strong, clear bands despite trying various optimizations. In this picture, I tried 6 sets of 3 different dilutions.
1st dilution: 105.75 ng/µL 2nd dilution: 52.88 ng/µL 3rd dilution: 26.44 ng/µL
- Using 2 µL of each dilution in a 20 µL PCR reaction.
Annealing temperature are;
- 1st 3 wells = 60°C (Wild Type of 1st gene)
- 4th, 5th and 6th = 62.3°C (Wild Type of 1st gene with Forward replaced by forward of Mutant)
- 7th, 8th and 9th = 56.3°C (Mutant of 1st gene)
- 10th, 11th and 12th = 56.3°C (Mutant of 1st gene with Forward replaced by forward of Wild Type)
- 13th, 14th and 15th = 50.3°C (Wild Type of 2nd gene)
- 16th , 17th and 18th = 47°C (Mutant of 2nd gene)
I've tried adjusting annealing temperatures, increasing the cycle number from 35 to 38, and increasing the extension time to 30 to 45 seconds but got no bands at all.
Any insights on optimizing my PCR conditions to achieve stronger bands would be greatly appreciated. Thank you!
I think that you may be using too much dna with some pcr inhibitors present.
Try the pcr with 13 and 6 ng dilutions. Diluting the inhibitor may lead to increased yield even though there is less dna.
I agree with Paul Rutland , you should dilute out the DNA.
What size products are you expecting? Sorry if it's in the picture, but the image isn't loading right on my monitor. Rule is 1 minute per 1 kb for most polymerases.
One thing that can often help is to toss out your primers & make new ones from the stock. Primers degrade over time, especially with repeated freeze-thaw cycles or long-term storage in 4*C
Good luck!
You can adjust the concentrations of MgCl2, dNTPs, primers, and Taq polymerase. In some cases, adding bovine serum albumin may also improve results.
Hi Everyone, Yes I diluted the DNA with 13 and 6 ng dilutions as suggested but from all these sets, I got only one band (last well from previous image) from 13 ng dilution. It's amplicon size is 460 bp which is my desired size and rhe band was good as well. But didn't get any other bands even the faint ones like I got previously in the picture I shared. They should be at 551 and 518 bp.
For better quality band you can check the amount of DNA in your sample. the ideal Nano Drop ration for DNA is 1.5-1.8, if it give the same ratio, than move to check the accessories like agarose, the buffer, etc. if these Ok, than move to primer concentration (10 or 16 micromolar), and finally check the annealing temp, just set it one degree above and below from the given temp.
U can adjust PCR reaction conditions i.e increasing the extension time and number of cycles.
1.Reduce your template – Having too much of a template seems to be the most common problem. Attempt to diminish your template in order to assess if that has an effect on your results. 2. Lower cycle rates – This is of special importance when your template's concentration is higher. Maintain between 20-35 cycles. 3.Reduce the length of the extension process / Increase the temperature of the annealing process - both of these will have the effect of improving your results from PCR by decreasing the frequency of non-specific binding and streaking bands. For small gels, make sure you're changing your TAE every time you run. It's logical to assume that larger rigs can have several runs without being replenished. However, this could be a problem for you.
Dear Maira Zafar
which is the expected size of your gene?
it seems to me that you have some weak band just in the line 1-5 those are the lines with the higher annealing temperature. I think you can try to:
1) Increase a little the annealing tempeature up to 65?
2) Try a different Taq polimesare
best regards
Manuele
- Badges
- Science method
- Similar topics
- Biological Science
- Ecology