
Blunt-cloning -all are vector self-ligation products?
#16
Posted 23 March 2005 - 02:47 AM
The vecotr insert ration should be atleasr 1:3
#17
Posted 18 February 2009 - 07:16 AM
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.
#18
Posted 25 February 2009 - 08:26 PM
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...
#19
Posted 04 March 2009 - 04:16 AM
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?
Our country has a serious deficiency in lighthouses. I assume the main reason is that we have no sea.
I never trust anything that can't be doubted.
'Normal' is a dryer setting. - Elizabeth Moon
#20
Posted 04 March 2009 - 04:30 AM
if this is a PCR product, using of a proof reading polymerase would create blunt end products.
#21
Posted 04 March 2009 - 06:03 AM
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.
Our country has a serious deficiency in lighthouses. I assume the main reason is that we have no sea.
I never trust anything that can't be doubted.
'Normal' is a dryer setting. - Elizabeth Moon
#22
Posted 04 March 2009 - 07:52 AM
talent does what it can
genius does what it must
i used to do what i got paid to do
#23
Posted 04 March 2009 - 10:35 PM
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.
#24
Posted 06 February 2010 - 06:38 PM
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
#25
Posted 07 February 2010 - 04:44 AM
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.
#26
Posted 25 March 2010 - 02:20 AM
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.
#27
Posted 21 April 2010 - 10:16 PM
http://www.flashpapers.com
#28
Posted 23 January 2011 - 01:23 AM
#29
Posted 23 January 2011 - 07:41 PM
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.
#30
Posted 05 June 2012 - 09:45 AM