3 replies to this topic

[Ambinlab]

Hello

Pleassseeeeeeeeeeeeee is there anybody who help me to start with lentivirus, siRNA,shRNA and knocking down the genes?

I just begin working in molecular biology lab and my colleagues just gave me some 500pages ebooks to read!!!

Thanks in advance

Dreamer

[LostintheLab]

Don't panic! First off.

If you're not so familiar with these techniques, do some background reading- text book, introduction of those ebooks, some general internet searches or pubmed for a review article. Just so you know what the principles are.
I guess you'll be designing these to knockdown a particular gene, so you'll need to know the sequences, maybe restriction enzymes, to help choose good vectors- there are plenty of these programes available for free on the internet to help you with that, they can even help find good siRNA targets or shRNA sequences.  Maybe you'll be using a kit for this. I use the invitrogen lentivirus kits, using the entry vector system which makes it easy to make a variety of different types of vectors- I know other companies have similar systems. They also have lots of help pages which are good to look at for more practical details.
Once you've read a bit more and have more specific questions- ask the guys in the lab, or ask here.

[Ambinlab]

LostintheLab, on 15 December 2010 - 04:49 PM, said:

Don't panic! First off.

If you're not so familiar with these techniques, do some background reading- text book, introduction of those ebooks, some general internet searches or pubmed for a review article. Just so you know what the principles are.
I guess you'll be designing these to knockdown a particular gene, so you'll need to know the sequences, maybe restriction enzymes, to help choose good vectors- there are plenty of these programes available for free on the internet to help you with that, they can even help find good siRNA targets or shRNA sequences.  Maybe you'll be using a kit for this. I use the invitrogen lentivirus kits, using the entry vector system which makes it easy to make a variety of different types of vectors- I know other companies have similar systems. They also have lots of help pages which are good to look at for more practical details.
Once you've read a bit more and have more specific questions- ask the guys in the lab, or ask here.

Thanks I am really overwhelmed:(
I am reading about all these techniques but the probelm is that I cant find a book which tell me the whole process step by step. eg, I read about packaging and then read about t293s and then titeration of virus and I donot know where to start....

Any idea?

[LostintheLab]

the packaging part- is just the vectors you need to add to make the virus. I've used the invitrogen lentivirus kit, their protocols are step by step- maybe you should look there if you want a step by step guide to start you off (or what ever virus system you will go with). The packaging part is just a simple transfection. The vectors are added to the 293 cells and the virus is made and released into the supernatent which you collect.
Then comes the titering- another cell line is useful for this here, but if you read you'll see the choices they suggest. If you are selecting with antibiotics you'll need to work out the best concentration for killing cells here before you set up the titre. Titering is easy enough- just a serial dilution of the virus containing supernatent and then after selecting with the antibiotic you count the remaining cell colonies to get an idea of how much virus you've made.
Just break it down in parts. The first part- making the vectors with the right sequences and setting up the transfection to make the virus is the first part, so concentrate on that. The titering comes after and the supernatents can be frozen in the -80C until then.