Cell

Part:BBa_K300984:Experience

Designed by: Manuel Lupotto, Lorenzo Pasotti, Paolo Magni   Group: iGEM10_UNIPV-Pavia   (2010-10-09)

This experience page is provided so that any user may enter their experience using this part.
Please enter how you used this part and how it worked out.

Applications of BBa_K300984

User Reviews

UNIQe63f18ea6e492b22-partinfo-00000000-QINU

••••

UNIPV-Pavia iGEM 2010

This E. coli strain has been successfully used to propagate plasmids with R6K conditional replication origin.


BW25141 competent cells have been prepared according to a slightly modified version of the simple CaCl2 method (Sambrook J, Fritsch EF, and Maniatis T (1989), Molecular cloning: a laboratory manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.), yielding 10^5 CFU/ug of DNA after bacterial transformation of a pSB*** series vector plated on Cm.

Briefly, cells were grown to and OD600 of ~0.4-0.6, harvested (4000 rpm, 10 min, 4°C) and the supernatant discarded. Cells were resuspended in (30 ml for each 50 ml of initial culture) pre-chilled Mg-Ca buffer (80 mM MgCl2, 20 mM CaCl2), centrifuged as before and the supernatant discarded. Cells were resuspended in (2 ml for each 50 ml of initial culture) pre-chilled Ca buffer (100 mM CaCl2, 15% glycerol), aliquoted in 0.5 ml tubes and freezed immediately at -80°C. Test the transformation efficiency in Colony Forming Units (CFU)/ug of transformed DNA.


The same transformation efficiency was estimated after bacterial transformation of a R6K control plasmid (BBa_K300008 cut with XbaI-SpeI and self-ligated to generate a Cm-resistant R6K plasmid) plated of Cm, while the control strains (MC1061 and DH5alpha) yielded no colonies as expected because they cannot propagate R6K.


Miniprep of the transformed strains yielded a DNA concentration of ~20 ng/ul (qualitatively comparable with medium copy number plasmids).

UNIQe63f18ea6e492b22-partinfo-00000003-QINU