3' UTR of my gene of interests spans in a total of around 120 bp, although the 5' UTR is huge in kbs.
I am not able to design suitable primers to clone the whole UTR for luc-assay.
Tried making primers into the cds and genomic region around the UTR region. No positive results yet so far.
Any other ideas for cloning my UTR.
Will such short UTR show miRNA regulation?
Presumably if you know this much about it, you know the sequence. Why not just get the UTR sequence synthesised? For such a short sequence, this shouldn't cost too much (certainly cheaper in overall cost when weighed up against the cloning, PCR and other reagent costs, plus your time).
And yes, 120bp is still within the realms of miR regulation.
Get first strand cDNA sythesised with Tailed Oligo (dT) primer. Now use this first strand cDNA mix to amplify your full-length transcript using forward gene specific and an anchored sequence binding reverse primer to amplify the desired full-length transcript. You can use this as template to amplify again with a reverse primer binding before poly A-tailing site. However, you can use the poly A tailed product too for cloning.
How are you going about primer design? Is this an A-T or G-C rich region?
@heather my reverse primer (3'-5') is lying in poly A tail region, if i utilize cDNA as a template. To avoid this, i have tried to utilize the genomic DNA as a template to avoid multiple AT rich regions for my reverse primer binding. No positive results yet so far.
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