Blunt-cloning -all are vector self-ligation products? - (Jul/11/2003 )
hi, karen.....
besides the previous answers, CIP treatment and proper purification of both, vector and insert, take care on the ligase buffer...after several defrozing steps, ATP could be depeleted. I improved results adding ATP 10 mM to ligase reactions. If the buffer is not so old, aliquote it in small amounts, just to use once or twice.
rgrds
To stimulate the blunt-end reaction, 150-200mM NaCl and 5% PEG can be added. 
As above, but you can either dephosphorylate your vector with SAP, or phosphorylate with TAP and T4 polynucleotide kinase.
Hi...Karen. Have you solved your problem?
There are some factors that influence result of Blunt-end ligation, including de-phosphorilation of vector, vector purification after de-phosphorilation etc. As usual ligation, you also should pay attention on ration of insert and vector concentration. In my experience, BAP (Bacterial Alkaline Phosphatase) also work well for de-phosphorilation of vector.
Good luck
.after several defrozing steps, ATP could be depeleted. I improved results adding ATP 10 mM to ligase reactions. If the buffer is not so old, aliquote it in small amounts, just to use once or twice
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
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
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.
InvisibleSurfer on Jul 27 2004, 03:20 AM said:
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...
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?
this is perhaps a stupid answer,
if this is a PCR product, using of a proof reading polymerase would create blunt end products.