Cloning into lentiviral vector - (Jul/08/2011 )

Dear All,

I have been trying to clone my insert (1.1kb) into a lentiviral vector (10 kb). I never have any colonies on my control plate (ligation without insert) and I do have some colonies (5-20) in my plate. However, when I do dnaprep and digest with the same enzyme, I cannot release any insert from them.

Please help. I need to finish this as soon as possible

Hello?
Is there anybody who can give me some suggestions about this issue?

what enzymes are you cutting with? single or double digest? what do you dephosphorylate with? I use antarctic phosphatase as you can deactivate that by heating to 60C.

Then clean, I prefer to phenol/chloroform rather than column.

what are your ligation conditions? you are getting self-ligating colonies rather than the ones with the insert, do you use a 3:1 molar ratio?

I use SpeI single digestion. I also use antarctic to dephosphorylate. I use column though. My ligation conditions: room temperature for one hour using 3:! molar ratio.
I got only self-ligating colonies on my plate. However on the control plate (the one ligated without adding insert) I dont get any colonies.

have you tried different molar ratios?

No...basically this is the best ratio isnt it?

not always. i use 3:1, 3:2, 6:1

Thanks I will give it a try...

Regards

You can also try to increase the incubation time of your ligation (I use at least 5 hours).

And when working with lentiviral vectors, it is better to incubate the plates/mini prep cultures at 30ºC to avoid recombination (unless you are using the Stbl3 strain).

Good luck.

Hi,

Thanks for the valuable comments. I tried 3:1 3:2 and 6:1 ratios. Actually 3:2 and 6:1 ratios got me fewer colonies!!

I have not tried longer incubation hours yet. I use Mach1 competent cells. Is that OK for such a ligation?

thanks