I am working in RPA for the first time. I followed the protocol to design the primer using the twistdx manual. I bought target, which was fragmented. I figured out the exact sequence by using a PCR reaction where I used an RPA primer set. Now, I am not getting an intense band for 1e3, 1e2, 1e1, and 1e0 copies. Sometimes, I got some intense band with no template control. The weird band comes from protein and DNA binding.
I think the primer design is correct because I checked them by PCR reactions to verify the DNA sequence and RPA primers. My design was based on 30 nt,
Hello, dear Md. Ahasan Ahamed
There are some possible reasons for these kinds of bands, which are so-called "smear bands":
1. DNA degradation, contamination, or high concentration
2. Primer contamination, or high concentration
3. Wrong annealing temperature
4. If you use a specific DNA isolation kit, perform each washing step carefully. Sometimes a contaminated template DNA leads to smear bands.
5. Reduce your PCR cycles
6. Decrease the amount of PCR product you load on the gel
And to obtain more intense bands, you can:
1. Load a high concentration of templates.
2. Increase your PCR cycles
3. Load more amount of the product on the gel.
Hello Mohammad Ali,
Can you please list some aspects of RPA reaction? You informed me about the PCR reaction. I am working with RPA reaction (Recombinase Polymerase Amplification)
you can also use DMSO to increase linearity of your template (at 5% of the total volume), but first check for quantity and quality of your samples, they must be homogenous.
all the best
fred
Hi Fred,
Should I add 5% DMSO before gel? Can you tell me the name of commercial product and protocol?
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