Hi everyone,
I'm planning to do shRNA cloning in the pLKO.1 for lentivirus production. i read the protocol provided by addgene et some others. There is a little that I don't understand regarding the Age1 restriction site. The protocol is based on annealed oligos that you put the open vector. The bases recommended to add at the 5' side of the forward oligo are CCGG. but the restriction site normally is CCGGT. Why we don't put the "T" at the end of this site whereas they put a full site for EcoR1? Hope i was clear . thanks in advance
Hi there,
The cloning strategy is based on using AgeI site only for ligation. pLKO1 is originally containing ash RNA which is extracted by AgeI/EcoRI double digest. As the first screen is based on EcoRI/NcoI double digest, the conservation of AgeI site is not necessary. In fact it's even better to lose the site during successful ligation: plasmid unsensitive to AgeI will be positive for the expected insertion.
In case you need more details, go to Addgene site:
http://www.addgene.org/tools/protocols/plko/#C
Chaveroux,
I am not sure if this will answer your questions, but I've cloned multiple shRNAs into the pLKO.1 (AgeI/EcoRI). What I do is setup the oligos as such: (random sites at 5' to be cut later on, followed by AgeI 5', sense, loop, antisense, EcoRI 3', Terminal Ts, Enzyme random sites 3').
I've added an attachment for an example shRNA that I have previously used.
As seen in the attachment, you want to order the oligos in such a way that they will be complementary in the middle (~10bps). You can then perform a 30 min klenow reaction to extend "into complete double strands", follow by AgeI/EcoRI digest, and subsequent ligations into pLKO.1. I use a 1:10 (oligo:vector) ratio and I get tons of positive clones.
http://journal.frontiersin.org/article/10.3389/fimmu.2015.00141/abstract
Hope that helps
Mahmood
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