Blunt-cloning -all are vector self-ligation products?
Posted 11 July 2003 - 06:35 AM
Posted 11 August 2003 - 10:05 AM
Posted 29 August 2003 - 09:57 AM
Serge Champetier Ph.D.
Posted 11 September 2003 - 12:17 PM
Posted 22 September 2003 - 07:23 PM
yes it works very well in our experiment,too.
and i add the PEG4000 for the ligation.
Posted 09 October 2003 - 09:55 AM
And, I use CIP to dephosphorylate the vector, but I found that it's quite difficult to inactivate it before ligation.
Posted 23 October 2003 - 11:48 PM
not use PEG4000.
it will give you well ligation.
Posted 08 March 2004 - 12:02 AM
I subcloned a CYP3A4 cDNA into pShuttle vector (Clonetech) using blunt-end cloning method. I use DraI (a blunt-end enzyme) to digest pShuttle and phosphatase using CIP. The insert was cut and then fill in at 5'end using Klenow. It is almost empty on the control plate of vetor itself with ligase, and almost hundreds of clones on the vector and insert plate. But when I selected around 50 clones using restriction digestion or bacteria PCR reaction, not one xlone containing insert. Why?
I use to have same problem. Do you recover your insert fragment from agarose gel then treat a fragment with Klenow? Please check that phenol is removed completely before treat with Klenow by extraction with chloroform again. I also use SAP. Good luck.
Posted 27 July 2004 - 03:20 AM
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
Posted 15 August 2004 - 12:13 AM
Inaccordance to your elucidation, it may be the poor efficiency of dephosphalation.
So I propose you to change the kit , SAP is really good and easy to inactive before ligation entry.
If your vector has the Lac screening marker , do a white-blue try.
Edited by sunrise, 15 August 2004 - 12:13 AM.
Posted 18 August 2004 - 05:35 AM
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.
Posted 08 September 2004 - 05:00 AM
Posted 12 October 2004 - 05:17 AM
Posted 29 November 2004 - 04:46 AM
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.
Posted 02 January 2005 - 07:39 PM
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?