Our team had been trying the procedure that is outlined in "Direct PCR: a new pharmaceutical approach for the inexpensive testing of HLA-B* 57:01" study to detect HLA-B gene on HIV patient samples with a in house method, but i have a question.
Whenever we did the gel electrophoresis for the First step PCR and Second step PCR samples, we did not observe any LOXL gene at 444 bp for our negative samples.
A few times we got lucky and we were able to see the LOXL indicator gene, but when we looked at the nested PCR gel electrophoresis result for the same sample, we couldn't see any LOXL internal control genes.. Do you by any chance know the reason why we can't get a consistent result for our negative samples?
We have checked our temperatures, material qualities including our primers etc. Thank you.
i think but not confirm that these conditions happens when the samples not completely pure specific, so first of all i think you must firstly be sure from your DNA sample purity.
second choice is to optimise the samples concentrations as it may be the cause if you dont mix your DNA sample well so concentrations may vary.
finally you can increase your first step PCR one or twwo additional (more) cycles and so obtain more no. of copies inter the next PCR step (2nd PCR).
I hope these answers can help??? and am waiting and following other answers by researchers
Dear Rukiye,
It is always a little bit difficult to make PCRs with four or more primers.
(It could take a long time to establish a new multiplex PCR system!)
In every PCR system you could need a little bit different primer concentrations.
I would guess, that you take primer concentrations from this publication.
In this case you should vary your primer concentrations in your system.
Perhaps you would need higher concentrations for the primers of your control amplfikat?
Best reagrds,
Thomas
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