High-throughput cloning and expression in Lactococcus lactis
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Table 1 and 2
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.
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
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.
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
Posted 25 Jul, 2007
High-throughput cloning and expression in Lactococcus lactis
Posted 25 Jul, 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.
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
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.
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
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