8 replies to this topic

[prabhubct]

Does anyone trying to treat  vector cut salI treatment with Calf alkaline phosphate and Ligating it with insert cut at end with salI. I finding problem of no colony in ligation. Suggestion Welcome. Thanks.

[pDNA]

possibly your insert is not cut properly (e.g. SalI has problems cutting PCR products).

I would check if self ligation of your insert is possible by loading a ligation reaction that only contains the insert + ligase on a gel ...should give you multimers of the size of the insert (like a ladder).

Regards,
p

[prabhubct]

Thanks pDNA,
          I appreciate your suggestion. About PCR product with SalI site I found really much difficult so I cloned it in Blunt end cloning vector and then cut it with SalI. But Now insert cut with SalI from vector I am finding difficult to clone in SalI vector treated with Calf alkaline phosphatase. I checked for vector with Calf ALP treated for self ligate- No colony (Negative control).

[phage434]

Have you tried the ligation without CIP treatment?  My guess is that the treatment is trashing the DNA ends, and inhibiting ligation.  I would do the ligation without CIP at low concentrations of vector and insert (10 ng of vector DNA in a 10 ul reaction, equimolar amounts of insert).  Do you have an easy way to identify correct clones? Your competent cells could also be a problem.

[prabhubct]

I would have liked to do as you say. But I don't have selection marker ( e.g.blue -white screening)  for identifying  clone positive for insert. No CIP treatment would lead simply to self ligate. There are chances that I may get ligated product vector too. How can I identify that out of so many colonies.

[phage434]

If you need to dephosphorylate your vector, you should do it with the minimal amount of CIP and for the shortest time possible.  Switching to shrimp alkaline phosphatase or antarctic phosphatase would be a good idea, so that you can heat inactivate it.  Another option is to use PCR to amplify your vector backbone, then treat with DpnI to get rid of parent circular vector.  The PCR product will not transform (ideally, you would use different restriction enzymes at both ends, to eliminate the recircularization after cutting and ligating).

You didn't say how you know your cells are competent (and, equally important, how competent).

[prabhubct]

Thank You Phage434. I like your suggested PCR amplify approach. For now I am planning to use Shrimp alkaline phosphatase.I wonder if there is overlapping PCR amplification approach somebody suggest, I just have rough Idea that this could be possible way.


         I have checked whether cells are competent by transforming cells with PUC18, they shows colonies. I did not check for transformation efficiency.

Edited by prabhubct, 06 August 2012 - 10:35 PM.

[prabhubct]

phage434, on 03 August 2012 - 11:01 AM, said:

If you need to dephosphorylate your vector, you should do it with the minimal amount of CIP and for the shortest time possible.  Switching to shrimp alkaline phosphatase or antarctic phosphatase would be a good idea, so that you can heat inactivate it.  Another option is to use PCR to amplify your vector backbone, then treat with DpnI to get rid of parent circular vector.  The PCR product will not transform (ideally, you would use different restriction enzymes at both ends, to eliminate the recircularization after cutting and ligating).

You didn't say how you know your cells are competent (and, equally important, how competent).

Efficiency check of competent cell prepared by TSS protocol:-

1.   Added 0.1 ng of PUC 18 in 100 microlit competent cells
2.   cool on ice 10 min, room temp 10 min, ice cool 10 min,
3.   LB 900ml added, incubate 37 ⁰C for 1 hr.
4.   Centrifuge 4000rpm / 5 min. to pellet bacteria
5.   Dissolve pellet in 100 microlit LB
6.   Take 10 microlit of above (step 5) and add 990 ml LB - not incubated
7.    Plate 100 microlit of above on ampicillin resistant LB plate, incubate 37 ⁰C O/N


As per this protocol I have used 1 pg PUC 18 to check transformation efficiency.
Protocol link http://www.sigmaaldr...efficiency.html

I did not get even single colony.

When I used 100 ng of vector I got 20 colonies.

[phage434]

Well, that's your problem.  Your efficiency is 20/0.1 = 200 cfu/ug, which is very very far away from the good efficiencies of 10^8 to 10^9 cfu/ug, and actually much worse than CaCl2 transformation, which usually comes in at about 10^6 cfu/ug.

You might want to try this:
http://openwetware.o...competent_cells