I'm working on the viral chitinase gene. But I'm having trouble with PCR.
I can't get the results I want.
PCR product to clean the gel and use the same product as a template to make PCR again, but in different places band out.
In sequence results, I find a series that has nothing to do with my gene sequence.
I couldn't get through this before.
What would you recommend about this?
Thanks
when you re amplify a pcr product every 10 cycles is 1000 times more product. If you run more than about 15 cycles in the second amplification then the amount of product is huge and it anneals to itself and just produces a long smear of amplified product which will not look much like the original amplimer. How many cycles of pcr re you running in both first and second rounds of pcr?. Also is your no template control clear (no product) in both pcrs?
Hi,
Are your peimers specific? Hits? It should be specific for the specie DNA that you are trying to amplify.
Are they self complementary?
Is ur product size huge?
##Reduce primers conc and PCR cycles.
Cutting from gel and amplyfying it will not solve the issue in this case, as it might not be the desired band.
hi
you should use primer sequence from high impact article .
1- you can used from standard virus strain or ready DNA or cDNA as positive control
( for example AcMNPV ChiA >> https://www.atcc.org/products/all/VR-2268.aspx )
https://www.atcc.org/products/all/VR-2268.aspx
2-Run PCR by high quality cDNA or DNA from (10-20ng in 25 ul total reaction PCR) . in this case you should set up your reaction by gradient annealing temperature .
2- If you get negative feedback with positive control, you need to redesign the sequence of primers.
3- If your ultimate goal is to produce a transgenic plant that is resistant to pests,you can order the desired sequence in the desired plasmid according to the preferred codon of the same plant for synthesis.
https://www.ebi.ac.uk/ena/data/view/CAD79454&display=fasta
good luck
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