When I optimized my designed primer on human standard DNA it worked and gives the selected exon/PCR product. but its using for subject samples few samples are getting PCR product and few samples are not getting. I used DMSO its worked for few exons with same temp when I optimized but few are not working even though showing Product band along with non-specific bands.
Few are showing single band which is unwanted band (instead of 1298 bp showing 790 bp like) .
Please help me to overcome this problem.
If my subject DNA is slightly problem, what can I do to amplify this?
We use DMSO as a PCR additive to increase the yield of a PCR reaction on GC-rich DNA templates. It increase yield by preventing the formation of secondary structures. But increasing the concentration of DMSO could also lead to decrease in PCR specificity. Your case might be due to the concentration of DMSO that you used because in different exon the G-C content may vary. I suggest you to reduce the concentration of DMSO. Or another alternative you can use Formamide instead of DMSO or you can use DMSO with BSA.
For more information, here I attach you one paper. This may be useful.
Dear Mr. Teketay,
When DMSO added PCR product was showing single good intensity band which was not mine. when I optimized my primers with human template standard DNA by without adding DMSO that was also superb.
Few genes of exons are easily polymerized by PCR with subject (patient) samples few are not polymerized so I used DMSO for polymerization few exons are getting product (from one subject sample with different exon regions) which I need and few exons are getting PCR product along with nonspecific bands and few exons are getting PCR Product instead of my product other exon was amplified ( showing band in different bp).
So could you give you a valuable suggestion for helping me.
Please find the attached gel pic DSC_7660 showing non specific bands, (Previously without using DMSO I got only single band by using human standard template DNA).
DSC_7674 Please check right side, that was 1.2kb but here showing 500bp change (Previously without using DMSO I got only single band by using human standard template DNA).
It doesn't sound to me like your fundamental problem is DMSO
- If some samples work under standard conditions and some do not that implies a sample specific issue. Have you checked the purity specs of your DNA ? Make sure your 260/280 ratio from spec or Nanodrop is >1.7 and your 260/230 ratio is >1.0
- For samples that fail to give products at all you could simply try a second round of purification; either with a 2nd column or Trizol if you are using manual extraction
- You also mention the fact that you have extra bands from some samples. This suggests that either your PCR annealing temp is too low or that your primers are not optimally designed. To render your PCR more stringent try increasing your annealing temp by 1-2C. This can sometimes eliminate a non specific band but retain your specific expected product
- With regards to primer design I am thinking that potentially your primer might be binding to a second site. Have you BLAST screened your primer to ensure it only binds with 100% accuracy to one site in your genome ?
Dear Laurence Sir,
Primers what I designed again I checked in BLAST, it gave 100% accuracy.
what you have given suggestion I will increase the annealing temp., but here, when I optimized my designed primers, that time those bands were good intensity. and few samples worked but few are not working because due to low conc. as per few one suggestion I added DMSO with previous annealing temp, PCR product gives my product along with nonspecific also.
Please suggest for this.
Thanks in advance
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