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How to propagate Electro-competent DB3.1 cells


Best Answer phage434, 18 August 2013 - 12:52 PM

Well, perhaps your DB3.1 strain has mutated its resistance gene. I believe Invitrogen no longer sells it, but we have some in the freezer if you'd like to try a different version.

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#1 Trys

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Posted 17 August 2013 - 03:01 PM

I ran out invitrogen DB3.1 stock, and want to make my own ccdb competent cells. Tried once, but efficiency for ccdb vector is extremely low despite high competency for pUC19.

Wonder whether I should add streptomycin to maintain genotype/ccdb resistance of DB3.1 due to its StrpR genotype.

 

Are there any other tricks to maintain ccdb resistance?

 

Thanks so much for advice,



#2 phage434

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Posted 17 August 2013 - 06:23 PM

We've never had this problem. I doubt streptomycin will help. An alternative to ccdB suppression of background is to PCR the vector, producing linear DNA that can then be cut with the correct enzymes (along with DpnI to eliminate template plasmid). Cloning with this dramatically reduces background.



#3 Trys

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Posted 18 August 2013 - 11:41 AM

Thanks phage434,

 

Good suggestions, please let me detail my goal here,

I am actually inserting a YFP PCR product into my destination vector that has ccdB and is 24kb long. But insetion site is not on ccdB sites, my goal is to use this insertion to modify my destination vector. But modfified vector has to reserve the ccdB sites for later LR reaction. 

 

However, I am stuck in this insertion ligation step and I highly suspect that my ccdB resistant DB3.1 cells cannot transform this either original circular destination vector or ligated vector since both contain ccdB sites.

 

Based on this background, I would like to hear further advice on how to make ccdB containing vector transformation work.

Thanks so much again,



#4 phage434

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Posted 18 August 2013 - 12:18 PM

Ah. Well, I'd suggest that the major problem might be the size of the vector, rather than the ccdB insert. It would be worth checking to see if the competence of your cells was high without the ccdB insert. I suspect that you will see similar transformation difficulties with such a large vector. Are you using electroporation or chemical transformation? If you prepare a large vector without ccdB, then you could also check the transformation efficiency of other strains, some of which are optimized for large vectors.  Have you thought of replacing the ccdB selection with a visible phenotype, such as LacZ (on S-Gal plates) or GFP/RFP proteins?



#5 Trys

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Posted 18 August 2013 - 12:42 PM

phage434,

 

Thanks very much again and would like to further your suggestions here,

 

Ah. Well, I'd suggest that the major problem might be the size of the vector, rather than the ccdB insert.

 

I thought potential size issue as well, I tested another ccdb vector (19kb) with inserts that replaced ccdb sites as a size control vector. Similarly, this 19kb vector with ccdb cannot transform DB3.1, but vector/insert without ccdb had very high tranformation, indicating high competence of the self-made DB3.1 cells.

 

It would be worth checking to see if the competence of your cells was high without the ccdB insert. I suspect that you will see similar transformation difficulties with such a large vector. Are you using electroporation or chemical transformation?

 

I made electro-DB3.1 competent cells.

 

If you prepare a large vector without ccdB, then you could also check the transformation efficiency of other strains, some of which are optimized for large vectors.  Have you thought of replacing the ccdB selection with a visible phenotype, such as LacZ (on S-Gal plates) or GFP/RFP proteins?

 

I would definitely need this ccdb sites for LR reaction, that is why my insertion modification is designed in places other than ccdb sites.

 

I appreciate your further insights based on the above discussion....



#6 phage434

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Posted 18 August 2013 - 12:52 PM   Best Answer

Well, perhaps your DB3.1 strain has mutated its resistance gene. I believe Invitrogen no longer sells it, but we have some in the freezer if you'd like to try a different version.



#7 Trys

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Posted 18 August 2013 - 01:44 PM

That will be very helpful, phage434, I truly appreciate,

could you suggest me how to get from you?  I am in California,

Thanks very much again,






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