Stanford/BIOE44:Module 1:Day3: Difference between revisions
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(New page: {{Template:Stanford/BioE44}} <div style="padding: 5px; width: 656px; border: 2px solid #397D02;"> ==Module 1: Day 3== ===Before Class=== *Please read:) |
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== | ==M1: Day 3 - Electrocompetent Cell Prep and Transformation== | ||
=== | ==Introduction== | ||
==In Class== | |||
====Electrocompetent Cell Prep==== | |||
''The first two steps of the protocol have been done for you.'' | |||
#Pick an isolated colony from an LB plate and grow overnight in 3–5 ml of LB at 37C. | |||
#Next morning, add 0.5 ml of the culture to 25 ml of LB in a 250-ml flask and grow at 37C to an OD600 of 0.50–0.60. | |||
#Transfer the culture to a 50-ml Falcon tube and spin at 6,000g in prechilled rotor for 10 min at 4C. | |||
#Wash the cell pellet with 20 ml of ice-cold H2O then centrifuge again as above. | |||
#Resuspend the pellet in 1 ml of H2O and transfer to a chilled 1.5-ml tube. Spin at 10,000g for 30 seconds at 4C. | |||
#Wash the cells again with 1 ml of ice cold H2O and centrifuge as above. | |||
#Repeat the above wash and spin step. | |||
#Resuspend the cell pellet in H2O in a final volume of 100μl and keep on ice. | |||
#Mix 0.5uL of your ligation reaction with 50μl of electrocompetent cells transfer into a 0.1-cm cuvette. | |||
#Introduce the DNA into the cells by electroporation (1.8 kV, 25 mF capacitance and 200 O resistance). | |||
#After electroporation, immediately add 1 ml of SOC and transfer to 1.5mL microcentrigure tube. | |||
#Incubate cells (with shaking or rotation) at 37C for at least 30minutes | |||
#Spin down cells for 2 min at 4000g in a microcentrifuge. | |||
#Resuspend the pellets in 200 μl of LB. Plate each aliquot of cells on a single LB plate containing the appropriate antibiotic. Grow for 12-16 hours (overnight) at 37C. |
Revision as of 22:57, 5 April 2010
M1: Day 3 - Electrocompetent Cell Prep and Transformation
Introduction
In Class
Electrocompetent Cell Prep
The first two steps of the protocol have been done for you.
- Pick an isolated colony from an LB plate and grow overnight in 3–5 ml of LB at 37C.
- Next morning, add 0.5 ml of the culture to 25 ml of LB in a 250-ml flask and grow at 37C to an OD600 of 0.50–0.60.
- Transfer the culture to a 50-ml Falcon tube and spin at 6,000g in prechilled rotor for 10 min at 4C.
- Wash the cell pellet with 20 ml of ice-cold H2O then centrifuge again as above.
- Resuspend the pellet in 1 ml of H2O and transfer to a chilled 1.5-ml tube. Spin at 10,000g for 30 seconds at 4C.
- Wash the cells again with 1 ml of ice cold H2O and centrifuge as above.
- Repeat the above wash and spin step.
- Resuspend the cell pellet in H2O in a final volume of 100μl and keep on ice.
- Mix 0.5uL of your ligation reaction with 50μl of electrocompetent cells transfer into a 0.1-cm cuvette.
- Introduce the DNA into the cells by electroporation (1.8 kV, 25 mF capacitance and 200 O resistance).
- After electroporation, immediately add 1 ml of SOC and transfer to 1.5mL microcentrigure tube.
- Incubate cells (with shaking or rotation) at 37C for at least 30minutes
- Spin down cells for 2 min at 4000g in a microcentrifuge.
- Resuspend the pellets in 200 μl of LB. Plate each aliquot of cells on a single LB plate containing the appropriate antibiotic. Grow for 12-16 hours (overnight) at 37C.