cloning of Poly A tail - (Sep/19/2006 )
Hi guys,
I am trying to introduce a Poly A tail sequence into the pBluescript vector digested with SmaI.I synthesized the Poly A tail by doing PCR using total cDNA from ovary. I cloned the PCR product into the vector and sent the clones for sequencing. But there was no Poly A tail in the clone confirmed by sequencing.Can anyone give me suggestions on how to introduce a poly A tail (of 50-100 bases long).
Thank you
Hello there,
By polyA tail, do you mean the signal that hooks on a polyA tail to a mRNA transcript during transcription or do you mean an actual run of As (ie AAAAAAAAAAAAAAAAAAAAA).
What tale might you have come to desire such a tail? I am just curious of what do you want to do with this poly A tail. I am being persumptuous but I have the feeling a run of As isn't what you are looking for.
BUt since I'm here, the fastest and simplest way to constuct such a run of mononucleotides is to purchase it as an oligo
Go to invitrogen or you usual company and purchase an oligo composed totally of a As. Invitrogen sells oligos up to 100bp long.
However I must caution, a blunt end ligation is difficult more so with something with such an AT content. In such a ligation, it would be better to hsve the oligo phosphorylated either as an extra with the synthesis company or by doing it yourself with PNK, definetly do an overnight ligation at 16 or even 4 Celcius. And finally must this polyA oligo go via blunt end ligation? Could another enzyme be used instead. Something with a sticky end.
Hi,
I mean a stretch of A's into my plasmid.Then I will do Invitro transcription by which I will get mRNA with Poly A tail added (for invitro transcription the antisense strand with Poly T will be used, thereby the mRNA will have A's).I am doing this way becuase I want to add defrinite length of Poly A tail to my mRNA. Otherwise I would have used the Poly A polymerase to add tthe A's at the end of my mRNA.I did order oligo from IDT. But they are taking time to synthesize it as it is not passing the quality control parameters.But thanks for the advice. I will phosphorylate the oligo before doing the ligation.
Thank you
By polyA tail, do you mean the signal that hooks on a polyA tail to a mRNA transcript during transcription or do you mean an actual run of As (ie AAAAAAAAAAAAAAAAAAAAA).
What tale might you have come to desire such a tail? I am just curious of what do you want to do with this poly A tail. I am being persumptuous but I have the feeling a run of As isn't what you are looking for.
BUt since I'm here, the fastest and simplest way to constuct such a run of mononucleotides is to purchase it as an oligo
Go to invitrogen or you usual company and purchase an oligo composed totally of a As. Invitrogen sells oligos up to 100bp long.
However I must caution, a blunt end ligation is difficult more so with something with such an AT content. In such a ligation, it would be better to hsve the oligo phosphorylated either as an extra with the synthesis company or by doing it yourself with PNK, definetly do an overnight ligation at 16 or even 4 Celcius. And finally must this polyA oligo go via blunt end ligation? Could another enzyme be used instead. Something with a sticky end.