cloning shRNA into pLentilox 3.7 - (Jun/22/2007 )
I am attempting to clone a 60bp shRNA into pLentilox 3.7. The construct that was made in this lab previously did not knock down the RNA. So I redesigned it (the loop sequence was actually inverted)
I am following this protocol to the letter: http://www.sciencegateway.org/protocols/le...rus/pllmap.html
My oligos were designed based on that protocol and are below:
5’ PTGG TGG TCA TGG CGC CCC GAA CTT CAA GAG AGT TCG GGG CGC CAT GAC CAC CTT TTT TC 3’
3’ ACC ACC AGT ACC GCG GGG CTT GAA GTT CTC TCA AGC CCC GCG GTA CTG GTG GAA AAA AGA GCTP 5’
The problem I am having is that I always get colonies on my plates after transformation, but they are never right. In fact, they arent even pLL 3.7. None of them are. I use a positive control digest (XbaI and NotI) of the original vector and I release the correct size band. I have tried this several times, with different concentrations of both plasmid and oligos.
One thing I should mention, is that I sequenced pLL 3.7 to ensure that it is 100% correct and there are several mutations in the U6 promotor, which either incorporates a stop site or deletes one. However, the maxi preps that use work and when I transfect 239t's, the cells are green. But, GFP is driven by another promoter and the shRNA is driven by the U6. But when I sequence the original plasmid (that doesnt knock down my gene of interest), I am able to get the sequence of the shRNA.
Does anyone have any suggestions? Is it possible that my oligos arent annealing properly? And why do I have this erroneous plasmid?
Sorry to inundate you all with questions-
Kelly
You might have to use cells which prevent recombination for the shRNA cloning. like SURE , Stbl3 etc. It can happen that you are losing the shRNA because of recombination.
As you write that there are mutations inthe U6 promoter, is it because of the modifications made ot the promoter or you got a different plasmid. verify the plasmid.
As you write that there are mutations inthe U6 promoter, is it because of the modifications made ot the promoter or you got a different plasmid. verify the plasmid.
Thanks for your help.
I am using Stbl2 cells. I looked up the SURE cells and it seems they may be more appropriate for this type of cloning (b/c of hairpin structure). What is your experience?
Yeah, I was thinking that the plasmid may be incorrect for one reason or another. I am in the process of ordering another from ATCC.
As you write that there are mutations inthe U6 promoter, is it because of the modifications made ot the promoter or you got a different plasmid. verify the plasmid.
Thanks for your help.
I am using Stbl2 cells. I looked up the SURE cells and it seems they may be more appropriate for this type of cloning (b/c of hairpin structure). What is your experience?
Yeah, I was thinking that the plasmid may be incorrect for one reason or another. I am in the process of ordering another from ATCC.
I have used SURE and Stbl3 cells for cloning shRNA into viral vectors. They are both good.
Good Luck !!!