I am trying to improve my PCR results by reducing smears in the product obtained from DNA extracted from dry tissue using the Norgen kit. The primers I am using have been successfully employed in previous publications to amplify the CoI mtDNA gene. To optimize the reaction, I have already tested variations in MgCl2 concentration, thermal cycling conditions, annealing temperatures, and the addition of enhancers such as BSA and DMSO. Fig 1 shows PCR amplification according the variation of the anneling temperature. PCR Master 8.7ng of DNA, 2.5 mM MgCl2, 0.20 mM of each dNTP, 0.1 µM of primers, 0.75 U of Taq Platinum. The thermal profile was 95 °C for 2 min, followed by 35 cycles at 95°C for 30 s, 49.5/50.5/51.5/53°C for 30 s, and 72 °C for 30”. And final extension step of 7 min at 72 °C.
Fig 2 shows variation of cycles. PCR setting was: 5ng of DNA, 3.3 mM MgCl2, 0.25 mM of each dNTP, 0.3 µM of primers, 0.5 U of Taq polimerase. Thermal profile was: 95 °C for 2 min, followed by 30/32/35/38 cycles at 95°C for 30 s, 49.5 °C for 30 s, and 72 °C for 30”. And final extension step of 5 min at 72 °C.
Fig 3 shows PCR amplification according the variation of the primers and the inclusion of DMS and BSA. PCR setting was: 5ng of DNA, 2.5 mM MgCl2, 0.25 mM of each dNTP, 0.2, 0.15, 0.1 µM of primers, 0.75 U of Taq polimerase, 5% DMSO, 1 mg/mL BSA. PCR was carried out under the following thermal profile: 94 °C for 2 min, followed by 35 cycles at 94°C for 30 s, 51.5 °C for 30 s, and 72 °C for 45”. And final extension step of 7 min at 72 °C. The sample 0.8 was run under the this thermal profile: 95 °C for 2 min, followed by 40 cycles at 94°C for 30 s, 51.5 °C for 30 s, and 72 °C for 30”. And final extension step of 5 min at 72 °C.
Could someone provide insights to improve my reaction?Thank you in advance
You are getting bands, which is great! Looks like the lower annealing temp is good. Most folks do 35 cycles, why are you bothering to test others? Standard primer concentration is 0.2 uMol, which is why that one looks the best.
The goal of PCR is to get bands the size you want, no need to optimize all of the parameters if it's working fine. Use 49.5* annealing, 35 cycles, 0.2 uMol primers.
I suspect you are loading way too much PCR product on your agarose gels, that is one reason you are seeing the streaking & the smearing bands. 5 microliters is usually plenty.
Your ladder bands aren't as even as they should be, most likely is from excessive salt. That tells me you need to wash your gel casting & running equipment (tray, combs, gel box, etc) in mild detergent and rinse very well with distilled water.
Good luck!
nested PCR. dilute the first PCR reaction 1:10 serially until you get 1:1000, 1:10k, 1:100k. then use it as template for a nested second PCR with primers that are inside of the first ones.
Dear Katie. Thank you for your helpful suggestions. I followed your advice and ensured better cleaning of the gel casting and running equipment, which resulted in improved band resolution. However, when I increased the PCR cycles to 35X and used a primer concentration of 0.2 µM, I noticed a significant increase in nonspecific bands. Unfortunately, this also resulted in the loss of amplification of the specific target band.
I’ve attached a picture of the gel for reference. I’d appreciate any further advice on optimizing these parameters to reduce nonspecific amplification while recovering the specific target band.
Best regards
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