11 replies to this topic

[Ambinlab]

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

[Ambinlab]

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

[PostDocTrauma]

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?

[Ambinlab]

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.

[PostDocTrauma]

have you tried different molar ratios?

[Ambinlab]

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

[PostDocTrauma]

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

[Ambinlab]

Thanks I will give it a try...

Regards

[leochicaybam]

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.

[Ambinlab]

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

[PostDocTrauma]

i've normally use STBL3 cells, but I'm not sure what the difference is, sorry.

[val80]

Hello!!!

I am trying to do something similar (ligation 12.4 kb vector and 6.7 kb insert), I am using DH5alpha and in the previous plates I got 4x colonies more in the ligation plates compared to the AP vector but they lack a piece...

This time I did ligation at 1:1 in 10 microliters volume with 50 ngr DNA total, 100 nanogram dna total and 1:2 ratio with 200 ng total... I have seen in other forums that 1:1 was the best ratio and different opinion in the total amount of DNA to be used...

Anyway, grow the cells at 37C for 12 hours then I saw that they should be better grown at 30C so I throw them at that temperature and this morning I got lots of colonies in the AP vector one and fewer colonies in the ligation one.. is this a good sign???