6 replies to this topic

[azada2z]

hi friends,
i've got a problem while working on PCR and clonings. please help me find a solution.

i am working on a virus (CMV) which has RNA genome. i need to find its nucleotide sequence.
usually i follow these steps-
1. RNA isolation from purified virus
2.cDNA synthesis
3.PCR amplification using Pfu pol(i get blunt ends but fidelity is high so that i get an authentic sequence)
4.A-addition @ 3'end using Taq pol(to get sticky ends with single Adenine added to the fragment)
5.ligation into T/A vector
6.transformation
7.plasmid isolation and confirmation of positive clone by PCR and double digestion before sending it for sequencing.

now the pTZ vector got over and I have no clue on how to clone my PCR fragments with limited resources. there are no restriction sites in my primers (degenerate primers), neither in the fragments (in other strains of CMV) that match the sites in other vectors we have.
please suggest me on how 2 go ahead.

[leelee]

redesign your primers with a restriction site on the end?

Just out of curiosity, what does CMV stand for with your virus? Is it that cucumber mosaic virus?

[HomeBrew]

Can you use the restriction sites that flank your insert in the TA vector?

[azada2z]

i cant order new primers now as i have to get the sequence of my PCR amplified product in hand and yes CMV stands for cucumber mosaic virus.
i would like to know what are the approaches to clone a (pfu)pcr fragments without T/A vectors. i checked for restriction sites in the terminal regions of my insert but it is not matching with any site in other vectors available in my lab.

Edited by azada2z, 27 July 2010 - 03:51 AM.

[ElHo]

You could try blunt end-cloning by omitting the tailing step of your PCR-product and digest one of your other vectors with a blunt-cutter.

[Adrian K]

what other vectors you have?

[perneseblue]

Sorry very off topic I was thinking CMV = member of cytomegalovirus family... and it became cucumber mosaic virus. That brought on a giggle.