I am using a PCR amplified product of around 56 bp with a T7 Promoter sequence and I am getting a single band after PCR. However, after in-vitro transcription of this PCR Product, I am getting two distinct bands on 15% Urea PAGE gel. I tried optimizing the reaction using Mg2+ and rNTP gradient. What should I do?
The presence of two distinct bands on a gel following in vitro transcription (IVT) of your PCR product suggests the existence of two distinct RNA species.
Couldn't that extra band be single-stranded or double-stranded DNA from the T7-PCR template ?
Do you make a DNase digestion, before loading your sample on a gel ? In order to digest the double-stranded DNA T7-PCR template ?
your sample may have been contaminated during the invitro transcription, as the presence of a double band signals the presence of two distinct RNAs
David Lepetit Yes, I performed DNase digestion of the in-vitro product before loading on the gel and I also loaded the template on the gel to compare. And the two bands are not those of the PCR product.
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