The gene I'm working on is HPPK-DHPS (folate). We isolated it from whole genome of seabuck thorn. So it has introns in it as well. Naturally the primers we designed were are against whole genome sequence of this gene. Now I'm done with the plasmid extraction and for its confirmation I had to run pcr. So far I have tried various annealing temperatures and have also used new kit of Takara, which is known for its efficiency. But still there is no result whatsoever. So much so there are not even dimers. As if today will be my 8th run of pcr. So long story cut short, I need to know what is it that I'm doing wrong?
Ghina,
Your question is not clearly worded. You talk about a plasmid 'huge in size' but is only 2.3 kb, 'isolated from whole genome' when plasmids replicate independently from the genome and are physically separate, on which you perform a PCR for a target you do not disclose, apparently using kits from Takara and Fermentas (for plasmid isolation? For PCR? We don't know), and apparently the PCR does not work.
Always Include a positive control with your experiments. It is a sanity check that will let you determine whether a negative result was caused by your experimental setup or your samples.
Thank you Oliver for the feed back. Yes I want to confirm whether there is a plasmid or not. I can most certainly check though with restriction digestion. It sounds helpful :)
Regards.
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