I m trying to amplified fragment of one betaretrovirus using primer which are designed on mouse sequence.
I amplified something but when I extracted band on gel and sequenced that i don't read the exact sequence.
Somebody can suggest me something to try to detected this sequence fragment?
thank you for help!
Are you trying to amplify a mouse betaretrovirus using primers designed on a mouse sequence? Or are you trying to amplify a betaretrovirus from another species using primers designed on a mouse sequence?
Are you doing a one step RT-PCR or a two step where you make cDNA prior to amplification?
Are you getting multiple bands on your agarose gel or just the single one? If the latter, then don't do a gel extraction but just purify your PCR product and send for sequencing.
What do you mean by don't read the exact sequence? Do you already know the sequence you should get or are you trying to identify the sequence from an unknown viral NA?
Hi Jhon,
sorry I write not correctly the answer. I m trying to amplied a human betaretrovirus that have an omology of sequence about 98% of the mouse. While, i m using primers designed on mouse sequence.
When I run on the agarose gel i see one band, i exctract that and sequencing. But I see only the primers and than i dont read the sequence that corrwsponds to mouse..
I don t know if i explicat well. I would Know another metods to amplified and to improvo this procedure.
Thank you
Did you obtain the expected size of PCR product on the gel?
Do you mean that you only got primer dimer by sequencing?
If you know the sequence of human betaretrovirus, why not design the primers targeting this sequence directly?
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