We are going to use SHC001 Scramble vector from sigma it does not contain any shRNA or GFP tag, however, it has ampicillin and puromycin resistance genes. In this case, how to characterize that transfection has been done.
Is there a protocol suggestion for transduction of e.coli with lentiviral particles. please suggest.
Hi,
Normally with the pLKO you can easily transform E.Coli and select the positive clones with ampicilin and then you prepare a MAXI. The protocols you can find easily in any kit.
Regarding infection or transfection in the target cells. For transient transfection, use PEI or lipofectamine. And for stable transfection (or infection), you would need the packaging plasmids to produce virus particles.
If you need a protocol for that let me know.
Best and good luck!
Hello everybody,
this protocol from Addgene is very useful for pLKO based lentiviral production. It includes everything from cloning to virus production and transduction.
http://www.addgene.org/tools/protocols/plko/
Hope it helps
Francesco
Hi Enrique,
So does it mean lipofectamine will not give a stable transfection with plko1? I have asked for the bacterial slabs of the vector and I am planning to isolate vector and then transfect it to cell line with lipofectamine will it work well? Is it mandatory to transfect cells with viral particles for stable transfection if yes then why? Please explain.
Please send me the protocol for viral transduction in bacteria.
Hi Heena, I use the same plasmid for my experiments, and I can give some pieces of advice: if you order the slabs from mission, plate on LB-ampicilin (I do plate on LB-carbenicilin), do a pre-culture, and then make a midi or maxi with a kit that is Endotoxin free, which is essential for the production of high-titer lentiviruses, so don't just rely on the amount of DNA you obtain. Additionaly, when you run out of maxi, you will have to re-transfrom, and this can be really painful, since you might not get any DNA, or get some contaminated DNA. To sort this out, it is better to use Stbl3-/- E.coli competent cells for re-transformation and maxiprep production.
To make a stable cell line, it is ideal to to do it by lentiviral trasduction. As Enrique said, you will need the packaging plamids (VSV-G, PAX2), HEK 293T (and not HEK 293) cells, and off course, your lab should be equiped with a S2-facility. For lentiviral transduction, there are several protocols, mainly depending on your target cell, so that you have to define as well. I do spin-infection (90 min centrifugation, 2200 rpm, 37°C) and it works quite well on my cells.
Good luck with experiments!
Sebastian
Hello everyone,
Is someone has idea If we transfect with lipofectamine how stable would be cell line?
Because we don't have 293T cells which are used for packaging.
Hello Heena,
the peack of efficiency will be at 72 hrs post transfection, then the plasmid will be lost. You can try to generate stable cell lines by using Puromycin selection (depending on which pLKO vector you picked). This will work but will take a lot more time, as very few cells will randomly integrate your plasmid (about 0.1 % - 1% indicatively). If you want to generate a stable cell line by transfection you should constantly apply Puromycin.
However I would recommend to find some 293t, they are quite common and I think any lab will share them gladly.
Hope it helps
Cheers
Francesco
thank you very much, Francesco,
Your suggestions were very valuable to us.
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