Can any one give me a detailed protocol for 16s rRNA PCR using 28F and 1492R primers. I want to know the concentration of working solution of the two primers and anneling temperature in particular.
HI Subhashish,
The detailed PCR reaction for 16s rRNA PCR amplification using 28F (One of the three) and 1492R primer is as follows:
1. 4 minutes, 94oC, 1 cycle
The amplification cycle is
2. 1 minute, 94oC, , 23 cycles (melting)
3. 30 seconds, 48oC, 23 cycles (Annealing)
4. 2 minutes, 72oC, 23 cycles (Extension for full length)
You can change the cycle number as per your convenience.
The optimal concentration of these primer is 200 nMolar. Annealing temperature of these primers are optimized between 48oC and 54oC.
HI Subhashish,
The detailed PCR reaction for 16s rRNA PCR amplification using 28F (One of the three) and 1492R primer is as follows:
1. 4 minutes, 94oC, 1 cycle
The amplification cycle is
2. 1 minute, 94oC, , 23 cycles (melting)
3. 30 seconds, 48oC, 23 cycles (Annealing)
4. 2 minutes, 72oC, 23 cycles (Extension for full length)
You can change the cycle number as per your convenience.
The optimal concentration of these primer is 200 nMolar. Annealing temperature of these primers are optimized between 48oC and 54oC.
Dear Shubhashish,
Use this concentration for ur master mix: template (200 ng, 2 µL); primers (1 µL, 20 µm); MyTaq Red Mix (25 µL) and sterile ddH2O (22 µL).
For your DNA amplification, use the following condition
initial denaturation of the template DNA (95°C for 5 min)
denaturation (95°C for 60 sec)
annealing (55°C for 60 sec)
extension (72°C for 1 min 30 sec)
and a final elongation (72°C for 7 min).
Set the process to 30 cycles and hold the system at 4°C until the PCR product is needed.
This condition should be okay for your primers
Would anyone have the qPCR protocol for primers 27F and 1492R?
Hi Shubhashish
The working primer concentration is 10 pmol per microlitre or 10 micromolar.
The PCR amplification conditions:
an initial denaturation step at 95 C for 5 min;
30 up to 35 amplification cycles of 30-s denaturation at 94 C;
plus 45 s annealing at 55 C;
plus 1.5-2 min elongation at 72 C and a final 10-min extension step at 72 C.
if you see any additional unwanted band, you can reduce amplification cycle to 28 cycle, also extention time could become shorter.
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