Jump to content

  • Log in with Facebook Log in with Twitter Log in with Windows Live Log In with Google      Sign In   
  • Create Account

Submit your paper to J Biol Methods today!
Photo
- - - - -

Blunt-cloning -all are vector self-ligation products?


  • This topic is locked This topic is locked
29 replies to this topic

#1 karen

karen

    member

  • Members
  • Pip
  • 1 posts
0
Neutral

Posted 11 July 2003 - 06:35 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?

#2 bobprashanth

bobprashanth

    member

  • Members
  • Pip
  • 3 posts
0
Neutral

Posted 11 August 2003 - 10:05 AM

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

#3 sergechampetier

sergechampetier

    member

  • Active Members
  • Pip
  • 8 posts
0
Neutral

Posted 29 August 2003 - 09:57 AM

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.

#4 ivpz

ivpz

    member

  • Members
  • Pip
  • 2 posts
0
Neutral

Posted 11 September 2003 - 12:17 PM

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

#5 lyrezxl

lyrezxl

    member

  • Active Members
  • Pip
  • 18 posts
0
Neutral

Posted 22 September 2003 - 07:23 PM

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

#6 carol855

carol855

    member

  • Members
  • Pip
  • 1 posts
0
Neutral

Posted 09 October 2003 - 09:55 AM

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.

#7 dragonboy

dragonboy

    member

  • Members
  • Pip
  • 2 posts
0
Neutral

Posted 23 October 2003 - 11:48 PM

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

#8 eyore

eyore

    member

  • Members
  • Pip
  • 2 posts
0
Neutral

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?

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. :rolleyes:

#9 InvisibleSurfer

InvisibleSurfer

    Enthusiast

  • Active Members
  • PipPipPipPipPip
  • 31 posts
0
Neutral

Posted 27 July 2004 - 03:20 AM

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

#10 sunrise

sunrise

    member

  • Members
  • Pip
  • 2 posts
0
Neutral

Posted 15 August 2004 - 12:13 AM

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! :P

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


#11 ale

ale

    member

  • Active Members
  • Pip
  • 20 posts
0
Neutral

Posted 18 August 2004 - 05:35 AM

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

#12 Sphingoman

Sphingoman

    member

  • Active Members
  • Pip
  • 6 posts
0
Neutral

Posted 08 September 2004 - 05:00 AM

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

#13 tuckern

tuckern

    member

  • Active Members
  • Pip
  • 18 posts
0
Neutral

Posted 12 October 2004 - 05:17 AM

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

#14 Ali

Ali

    member

  • Active Members
  • Pip
  • 15 posts
0
Neutral

Posted 29 November 2004 - 04:46 AM

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

#15 popogirlxd

popogirlxd

    member

  • Active Members
  • Pip
  • 19 posts
0
Neutral

Posted 02 January 2005 - 07:39 PM

[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




Home - About - Terms of Service - Privacy - Contact Us

©1999-2013 Protocol Online, All rights reserved.