29 replies to this topic

[sharath]

Generally ligases come with buffers for cohesive ends and also for blunt ends. So check out the buffer you use. Also, 10 times higher liagse quantity is advised for blunt end ligation.

The vecotr insert ration should be atleasr 1:3

[WHR]

Hi,
During or after ligation reaction, digestion with DraI can greatly reduce vector self-ligation. Just be sure that there is no DraI site in the insert DNA.

[wanie1985]

You don't necessarily have to use a blunt cutter; you can use ANY RE site, and then fill it in with Klenow. Alternatively, you can use dNTPs and Taq polymerase to fill the nt gap.

Way I did it (successfully):

- purify insert + vector with prefered method
- IF cutting with the same REs, mix plasmid + vector together and digest
- purify with prefered method
- add salt, dNTPs and Taq, incubate for a couple of hours
- clean up again
- add ligase + buffer etc and incubate o/n
- don't forget to pray between steps :-P

invisible surfer: may i know what is the temperature for incubation with Taq+dNTP?i'm having some trouble with ligating blunt end product as well. your method seems promising. i want to try with that approach...thank you very much...

[Trof]

I read a forum thread about how to get A overhangs from blunt products, but I need the oposite, to create a blunt and from products with A overhang.
We tried Mung bean nuclease, but that degrades the product moreover even in very small amounts of enzyme.

Has anyone have a functional protocol for blunting the products?

[perneseblue]

this is perhaps a stupid answer,

if this is a PCR product, using of a proof reading polymerase would create blunt end products.

[Trof]

If you trying to say that I could just add a proofreading polymerase to a finished PCR reaction that may be a bit stupid, but I wondered if someone has a protocol, how much enzyme for how long, same as the Taq treatment. Sure someone has it if it's that simple.

In any case what I meant is how to do it when I already HAVE my PCR product done with Taq polymerase that already makes A overhangs and THEN I want to blunt it. And for any reason I don't want to do the PCR with a proofreading polymerase instead.

[mdfenko]

you can use klenow fragment or t4 dna polymerase.

[WHR]

In any case what I meant is how to do it when I already HAVE my PCR product done with Taq polymerase that already makes A overhangs and THEN I want to blunt it. And for any reason I don't want to do the PCR with a proofreading polymerase instead.

Actually, you don,t need to do anything as Taq only add 3'A to small percentages of its products.

[novagen]

Hi all,

Iam new to this topic.
is it that only for blunt end cloning ,we have to dephosphorylate the vector . So, CIP treatment is not necessary for ligation of cohesive ends of vector and insert.

Thanx

[Vini]

Hi all,

Iam new to this topic.
is it that only for blunt end cloning ,we have to dephosphorylate the vector . So, CIP treatment is not necessary for ligation of cohesive ends of vector and insert.

Thanx

yeah, u r right. for cohesive ends, u do not require to give CIP treatment. In blunt-end it becomes necessary, to avoid self-ligation.

[hsb31]

Hi, Enthusiast
I am doing blunt-end ligation too and also have the trouble to get the clony. You suggestion is very good and I will try it. I am eagerly to know that why the ATP could be dpeleted after using several times. Could you tell me the answer?


Thanks

I think its because repeated freeze thaw degrades ATP.

[William Parkar]

Yes, SAP works very well in my experience. Definitely should dephosphrylate the vector before ligation.

http://www.flashpapers.com

[music_m3lody]

hi everyone..just drop by to ask a question regarding the ligase buffer. I know there are salts in the buffer (ready made buffer from company) but is it normal if we can see some precipitation in the buffer? it looks like salt precipitation. And my lab colleague didn't remember when they bought this ligase buffer.

[Kaioshin]

Yes, SAP works very well in my experience. Definitely should dephosphrylate the vector before ligation.

http://www.flashpapers.com

Warm it in your hand, maybe vortext it a little.
This happens frequently with NEB T4 Ligase buffer (10x) and it goes right back into solution.

[DaisyCarbine]

I've had good luck using antarctic phosphatase in blunt-ended ligation reactions -- you can heat-inactivate it at 65 C, so you don't have to purify the DNA before setting up your ligation rxn.