Dear colleagues,
When I used the old primer, I got dimer, I ordered a new primer, but I don't get any band in my product, please help me if possible.
Thank you for considering my question in advance.
Select a dna that is very well purified. It should have anOD260/280 of about 1.8.
If it is genomic dna then use 25NG of this dna and try amplifications with primers new forwards with new reverse, new forards with old reverse and new reverse with old forwars. Use an annealing temperature of 6c below the lowest Tm of these primers. Run 35 cycles.
Hopefully one primer set will amplify a band of the right size but with mny other non specific bands but once you have a primer set that works you can adjust the annealing temperature to make it more specific but it will be a good start to get some amplification
If you don't mind can you share primer sequence with gene name and also tell about which region you want to target..
Firstly you check the primer specificity with NCBI blast then go for wet lab
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