Team:Yale/Notebook/Week2
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<li>In the morning 4mL of the overnight 50mL culture was transferred to the 200mL flask. OD was checked ever 30 minutes. After 1 hour and 45 minutes the OD was at 0.588 (ideally should be 0.55). Cells were transferred to 6 sterile falcon 50mL polypropylene tubes (35mL put in each tube). Tubes were stored on ice for 10 minutes. Cells were centrifuged in Strobel lab upstairs at 2500xG for 10 minutes at 4C. Medium was poured off and tubes were stood upside down on paper towel + kip wipes for 1 minute to remove remaining medium. Pellets were resuspended in ice cold TB buffer. 10mL buffer was added to all 6 of the tubes to resuspend the cells. Then the tubes were combined to make 2x 30mL tubes. The two tubes were centrifuged at 2500xg for 10 minutes at 4C to recover the cells. Medium was poured out, and tubes were stood upside down on paper towel for 1 minute to remove remaining medium. Pellets were resuspended by gently pipetting in a total of 20mL of ice coldTB buffer (2x10 mL). Cells were combined in single tube. 1.5mL DMSO was added and tube was mixed by swirling. Tube was stored on ice for 10 minute. 100 microlitre aliquots were put into chilled eppendorf tubes. Tubes were closed tightly and flash frozen in liquid nitrogen and stored in the -80 freezer to the bottom left.</li> | <li>In the morning 4mL of the overnight 50mL culture was transferred to the 200mL flask. OD was checked ever 30 minutes. After 1 hour and 45 minutes the OD was at 0.588 (ideally should be 0.55). Cells were transferred to 6 sterile falcon 50mL polypropylene tubes (35mL put in each tube). Tubes were stored on ice for 10 minutes. Cells were centrifuged in Strobel lab upstairs at 2500xG for 10 minutes at 4C. Medium was poured off and tubes were stood upside down on paper towel + kip wipes for 1 minute to remove remaining medium. Pellets were resuspended in ice cold TB buffer. 10mL buffer was added to all 6 of the tubes to resuspend the cells. Then the tubes were combined to make 2x 30mL tubes. The two tubes were centrifuged at 2500xg for 10 minutes at 4C to recover the cells. Medium was poured out, and tubes were stood upside down on paper towel for 1 minute to remove remaining medium. Pellets were resuspended by gently pipetting in a total of 20mL of ice coldTB buffer (2x10 mL). Cells were combined in single tube. 1.5mL DMSO was added and tube was mixed by swirling. Tube was stored on ice for 10 minute. 100 microlitre aliquots were put into chilled eppendorf tubes. Tubes were closed tightly and flash frozen in liquid nitrogen and stored in the -80 freezer to the bottom left.</li> | ||
<li>Two tubes were set aside after being flash frozen. Contamination was tested by plating untransformed cells on an Amp plate. Competence of cells was tested by transforming a "control" plasmid containing Amp. 1ul of plasmid was added to 50microlitres of cells in a chilled 14mL bacterial culture tube to look at competence, and 1ul of ddH2O was added to 50 microlitres of cells in a chilled 14mL bacterial culture tube to look at contamination. Tubes were incubated on ice for 30 minutes. Cells were heat pulsed in a 37 bath for 45 seconds, then put on ice for 2 minutes. 450microliters of LB buffer was added to each tube, and tubes were incubated at 37C for 1 hour while shaking. 100uL of each culture was spread on an Amp-LB/agar plate and was put in the 37C incubator. </li> | <li>Two tubes were set aside after being flash frozen. Contamination was tested by plating untransformed cells on an Amp plate. Competence of cells was tested by transforming a "control" plasmid containing Amp. 1ul of plasmid was added to 50microlitres of cells in a chilled 14mL bacterial culture tube to look at competence, and 1ul of ddH2O was added to 50 microlitres of cells in a chilled 14mL bacterial culture tube to look at contamination. Tubes were incubated on ice for 30 minutes. Cells were heat pulsed in a 37 bath for 45 seconds, then put on ice for 2 minutes. 450microliters of LB buffer was added to each tube, and tubes were incubated at 37C for 1 hour while shaking. 100uL of each culture was spread on an Amp-LB/agar plate and was put in the 37C incubator. </li> | ||
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Latest revision as of 18:21, 27 September 2011