Hi, we do assays using FAm labelled RNA, and I use such oligos to do my terminal transferase assay. We want to see addition of one nt by enzyme( so in short we want to see that a RNA primer has changed from 20 mer to 21 mer) I see a sharp band for 20 mer, but the band for 21 mer looks like a smear. Can anyone please help if they have ran urea PAGE gels fr short RNA oligos? What preacautions should be taken while making samples or gel to improve the band sharpness and to get rid of smear.
I cannot help with this exact question although I used to sequence with 40cm gels and one base differences do show in old fashioned sequencing gels although longer products look clearer as thermal peak broadening is less so adding a nonsense 5' tail to the oligo to make the product larger might help a little.If your institute has a sequencer then in genotyping mode an additional base shows easily using standard POP gels on any capillary sequencer and the main problem is analysing the data as oligos are below the lowest size standards sold commercially and sizing accurately is difficult but just seeing the larger band is possible although if you use too much oligo band spreading may obscure the plus one band, does the band looklike a smear because it IS a smear?
Can your extension continue past the next base? . If you are relying on ddNTP to terminate the reaction , for instance, then you may need manganese in the reaction mix or DDnTPs may behave more like dNTPs and allow read through in reaction mixes which just contain magnesium as the divalent ion present for the enzyme to work
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