This is a preprint, a preliminary version of a manuscript that has not completed peer review at a journal. Research Square does not conduct peer review prior to posting preprints. The posting of a preprint on this server should not be interpreted as an endorsement of its validity or suitability for dissemination as established information or for guiding clinical practice.
Method Article
High-throughput cloning and expression in Lactococcus lactis
This is a list of supplementary files associated with this preprint. Click to download.
- supplement0.pdf
Table 1 and 2
Du Lihui
commented on 09 October, 2007
I found after Electrotransformation of L. lactis, the incubated cells should at least diluted five to ten fold before plating them on the M17 agar medium, or there will be no transforms appear. The colonies appeared not until two to three days, much longer than 18 hrs. The electrocompetent L. lactis NZ9000 was prepared more or less the same to the author, except that I directly innoculate the GM17 medium to the 2% ~ 2.5% glycine SGGM17 without the 1% glycine transition. I harvested when OD600 = ~0.3, but the transformation efficiencies was very low. Can the author give any suggestions since you are very good at manipulation of this bacteria? Thanks. My e-mail is [email protected] Waiting for good news from you.
eric geertsma
commented on 21 October, 2007
Dear Du Lihui, The problems you experience in transforming Lactococcus lactis clearly illustrate the need for the VBEx system. For electrotransformations of normal cut&paste ligation mixtures it is very common that few colonies only appear after two or even three days of incubation. Your competent cells are probably okay (although it would be good to test them by transforming a L. lactis plasmid instead of a ligation mixture). Only when the VBEx procedure is applied or when intacts plasmids are transformed, colonies will appear after overnight (~18 hrs) incubation. The transformation efficiency for material produced by the VBEx procedure is generally that high that plating of aliquots of 50-100 ul (that is 5-10% of the material) suffices. To circumvent the troublesome direct cloning in L. lactis, I would suggest to switch to the VBEx procedure. Plasmid requests can be directed to Prof. Bert Poolman ([email protected]). Best regards, Eric Geertsma
Du Lihui
commented on 09 October, 2007
I found after Electrotransformation of L. lactis, the incubated cells should at least diluted five to ten fold before plating them on the M17 agar medium, or there will be no transforms appear. The colonies appeared not until two to three days, much longer than 18 hrs. The electrocompetent L. lactis NZ9000 was prepared more or less the same to the author, except that I directly innoculate the GM17 medium to the 2% ~ 2.5% glycine SGGM17 without the 1% glycine transition. I harvested when OD600 = ~0.3, but the transformation efficiencies was very low. Can the author give any suggestions since you are very good at manipulation of this bacteria? Thanks. My e-mail is [email protected] Waiting for good news from you.
eric geertsma
commented on 21 October, 2007
Dear Du Lihui, The problems you experience in transforming Lactococcus lactis clearly illustrate the need for the VBEx system. For electrotransformations of normal cut&paste ligation mixtures it is very common that few colonies only appear after two or even three days of incubation. Your competent cells are probably okay (although it would be good to test them by transforming a L. lactis plasmid instead of a ligation mixture). Only when the VBEx procedure is applied or when intacts plasmids are transformed, colonies will appear after overnight (~18 hrs) incubation. The transformation efficiency for material produced by the VBEx procedure is generally that high that plating of aliquots of 50-100 ul (that is 5-10% of the material) suffices. To circumvent the troublesome direct cloning in L. lactis, I would suggest to switch to the VBEx procedure. Plasmid requests can be directed to Prof. Bert Poolman ([email protected]). Best regards, Eric Geertsma
Badges
Prescreen
This manuscript passed our Prescreen assessment
Complete author information
Appropriate declaration statements
Potential risks to human health
Prescreen
History
CURRENT STATUS: Posted
Version 1
Posted 25 Jul, 2007
Community comments: 4
Metrics
Comments: 4
HTML Views: ...
Associated Publications
High-throughput cloning and expression in recalcitrant bacteria
by Eric R Geertsma & Bert Poolman
Nature (04 July, 2007)
Learn more about our company.
This is a preprint. It has not completed peer review.
Method Article
High-throughput cloning and expression in Lactococcus lactis
Badges
Prescreen
This manuscript passed our Prescreen assessment
Complete author information
Appropriate declaration statements
Potential risks to human health
Prescreen
History
CURRENT STATUS: Posted
Version 1
Posted 25 Jul, 2007
Community comments: 4
Metrics
Comments: 4
HTML Views: ...
Du Lihui
commented on 09 October, 2007
I found after Electrotransformation of L. lactis, the incubated cells should at least diluted five to ten fold before plating them on the M17 agar medium, or there will be no transforms appear. The colonies appeared not until two to three days, much longer than 18 hrs. The electrocompetent L. lactis NZ9000 was prepared more or less the same to the author, except that I directly innoculate the GM17 medium to the 2% ~ 2.5% glycine SGGM17 without the 1% glycine transition. I harvested when OD600 = ~0.3, but the transformation efficiencies was very low. Can the author give any suggestions since you are very good at manipulation of this bacteria? Thanks. My e-mail is [email protected] Waiting for good news from you.
eric geertsma
commented on 21 October, 2007
Dear Du Lihui, The problems you experience in transforming Lactococcus lactis clearly illustrate the need for the VBEx system. For electrotransformations of normal cut&paste ligation mixtures it is very common that few colonies only appear after two or even three days of incubation. Your competent cells are probably okay (although it would be good to test them by transforming a L. lactis plasmid instead of a ligation mixture). Only when the VBEx procedure is applied or when intacts plasmids are transformed, colonies will appear after overnight (~18 hrs) incubation. The transformation efficiency for material produced by the VBEx procedure is generally that high that plating of aliquots of 50-100 ul (that is 5-10% of the material) suffices. To circumvent the troublesome direct cloning in L. lactis, I would suggest to switch to the VBEx procedure. Plasmid requests can be directed to Prof. Bert Poolman ([email protected]). Best regards, Eric Geertsma
Du Lihui
commented on 09 October, 2007
I found after Electrotransformation of L. lactis, the incubated cells should at least diluted five to ten fold before plating them on the M17 agar medium, or there will be no transforms appear. The colonies appeared not until two to three days, much longer than 18 hrs. The electrocompetent L. lactis NZ9000 was prepared more or less the same to the author, except that I directly innoculate the GM17 medium to the 2% ~ 2.5% glycine SGGM17 without the 1% glycine transition. I harvested when OD600 = ~0.3, but the transformation efficiencies was very low. Can the author give any suggestions since you are very good at manipulation of this bacteria? Thanks. My e-mail is [email protected] Waiting for good news from you.
eric geertsma
commented on 21 October, 2007
Dear Du Lihui, The problems you experience in transforming Lactococcus lactis clearly illustrate the need for the VBEx system. For electrotransformations of normal cut&paste ligation mixtures it is very common that few colonies only appear after two or even three days of incubation. Your competent cells are probably okay (although it would be good to test them by transforming a L. lactis plasmid instead of a ligation mixture). Only when the VBEx procedure is applied or when intacts plasmids are transformed, colonies will appear after overnight (~18 hrs) incubation. The transformation efficiency for material produced by the VBEx procedure is generally that high that plating of aliquots of 50-100 ul (that is 5-10% of the material) suffices. To circumvent the troublesome direct cloning in L. lactis, I would suggest to switch to the VBEx procedure. Plasmid requests can be directed to Prof. Bert Poolman ([email protected]). Best regards, Eric Geertsma
Associated Publications
High-throughput cloning and expression in recalcitrant bacteria
by Eric R Geertsma & Bert Poolman
Nature (04 July, 2007)