28 replies to this topic

[karen]

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?

[bobprashanth]

since blunt ends ligation is hard job to carry on. why cant you try using PEG in ligation mix.

[sergechampetier]

I have no good explanation for your results, but perhaps your vector dephosphorylation is not so efficient and for some reason the vector only ligation did not work well (or transformed well). Anyway, perhaps you could try SAP (Shrimp Alkaline Phosphatase) from Roche, which I think is better than CIP.
Good luck.
Serge Champetier Ph.D.
Quebec City.

[ivpz]

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

[lyrezxl]

try SAP (Shrimp Alkaline Phosphatase) from Roche.
yes it works very well in our experiment,too.
and i add the PEG4000 for the ligation.

[carol855]

I have try PEG 1000 for ligation, but still no improvment.
And, I use CIP to dephosphorylate the vector, but I found that it's quite difficult to inactivate it before ligation.

[dragonboy]

try Shrimp Alkaline Phosphatase
not use PEG4000.
it will give you well ligation.

[eyore]

karen, on Jul 11 2003, 06:35 AM, said:

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?

Dear Karen,

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.

[InvisibleSurfer]

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

[sunrise]

Dear Karen:
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.
Good luck!

Edited by sunrise, 15 August 2004 - 12:13 AM.

[ale]

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

[Sphingoman]

To stimulate the blunt-end reaction, 150-200mM NaCl and 5% PEG can be added.

[tuckern]

As above, but you can either dephosphorylate your vector with SAP, or phosphorylate with TAP and T4 polynucleotide kinase.

[Ali]

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

[popogirlxd]

[QUOTE].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