Hello everyone,
I´m performing a nested PCR, for a 799 bp amplicon. I have already performed a ton of temperature gradients but I keep obtaining a large unspecified band. Tm of my primers are the following:
- Tm_outPrimer FWD = 65.9 °C
- Tm_outPrimer RV = 67.7 °C
- Tm_nestPrimer FWD = 65.9 °C
- Tm_nestPrimer RV = 83.2 °C (joined to T7 promotor)
Here I share with you the result of electrophoresis in agarose gel for a temperature gradient of the 2° PCR (nest primers), for 62, 63, 64 °C of annealing temperature. The parameters of the thermocycler are:
- step 1: 95°C / 2 min
- step 2: 95°C /15 s
- step 3: (62, 63, 64 °C) / 30 s
- step 4: 68°C / 35s (not >68°C because of the OneTaq Quick-Load DNA Polymerase)
- step 5: 5 m / 68°C
I am open to receiving any comment or suggestion about my protocol. Any way you can help is welcome.
What do you suggest?
PCR products of 799 bp have been obtained. If only the obtained band is cut out from the gel and amplified by the Nested PCR primer again using 1 pg of purified DNA, the target is assumed to be further amplified.
Alternatively, consider changing the DNA Polymerase for amplification.
I think that the detection is sufficient. Since I do not know the purpose of the further experiments, I cannot give accurate advice.
as the above commenter suggested, you can cut the gel, purify, and then redo another nested PCR.
I would also check the quality and conditions of your electrophoresis gel. the ladder also doesn't seem coorectly migrated. so check on that side too.
Does it really need to be 1 perfect band for your purposes? If this is for cloning then just gel purify the product and call it "works well enough."
If you really do want 1 band (say for diagnostic PCR) then you can try a few things
1. different polymerase (Vent is good, Pfx, Platinum taq)
2. adding some Mg to your reactions
3. running smaller volume reactions (20 ul), those change temp faster than larger volumes
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