PCR & Cloning
Posted 26 July 2010 - 10:05 PM
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
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
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.
Posted 26 July 2010 - 10:51 PM
Just out of curiosity, what does CMV stand for with your virus? Is it that cucumber mosaic virus?
Posted 27 July 2010 - 03:08 AM
Posted 27 July 2010 - 03:47 AM
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.
Posted 27 July 2010 - 03:53 AM
Posted 27 July 2010 - 07:45 AM
..."best of our knowledge, as far as we know this had never been reported before, though I can't possible read all the published journals on earth, but by perform thorough search in google, the keywords did not match any documents"...
"what doesn't kill you, makes you stronger"---Goddess Casandra reminds me to be strong
"It's all just DNA. Do it."---phage434
Posted 27 July 2010 - 05:22 PM