Team:ULaVerne Collab/notebook
PROTOCOLS
Transformation Protocol (E. coli)
1. Pre-chill microcentrifuge tube and thaw competent cells on ice.
2. Add 50uL of competent cells into the chilled microfuge tube.
3. Add 4uL of DNA into the microcentrifuge tube.
4. Incubate on ice on 30 minutes.
5. Heat shock at 42 degrees Celsius for 45 sec.
6. Incubate cells on ice for 2 minutes.
7. Add 200uL of SOC media into the microcentrifuge tube.
8.Incubate and shake at 37 degree Celsius for 1-2 hours.
9. Pipette 200uL of cells into LB plate with ampicillin resistance.
10. Incubate plate overnight at 37 degrees Celsius.
2. Add 50uL of competent cells into the chilled microfuge tube.
3. Add 4uL of DNA into the microcentrifuge tube.
4. Incubate on ice on 30 minutes.
5. Heat shock at 42 degrees Celsius for 45 sec.
6. Incubate cells on ice for 2 minutes.
7. Add 200uL of SOC media into the microcentrifuge tube.
8.Incubate and shake at 37 degree Celsius for 1-2 hours.
9. Pipette 200uL of cells into LB plate with ampicillin resistance.
10. Incubate plate overnight at 37 degrees Celsius.
Miniprep
1. Transfer 1.5mL of overnight bacterial cultures from conicol tubes to microcentrifuge tubes. Centrifuge at 8000rpm for 3 minutes at room temperature. Discard supernatant and repeat until all of the overnight bacterial culture is suspended.
2. Resuspend pelleted bacterial cells in 250uL buffer P1.
3. Add 250uL buffer P2 and mix by inverting 4-6 times.
4. Add 350uL buffer N3 and mix by inverting.
5. Centrifuge for 10 minutes at 1300rpm.
6. Pipette the supernatant from step 5 to the QIAprep spin column. Centrifuge for 1 minute and discard flow through.
7. Wash the QIAprep spin column by adding 750uL buffer PE. Centrifuge for 1 minute and discard flow through.
8. Place the QIAprep column in a clean 1.5mL microcentrifuge tube. Add 50uL nuclease free water to the center of the QIAprep spin column. Let it stand for 1 minute. Centrifuge for 1 minute.
2. Resuspend pelleted bacterial cells in 250uL buffer P1.
3. Add 250uL buffer P2 and mix by inverting 4-6 times.
4. Add 350uL buffer N3 and mix by inverting.
5. Centrifuge for 10 minutes at 1300rpm.
6. Pipette the supernatant from step 5 to the QIAprep spin column. Centrifuge for 1 minute and discard flow through.
7. Wash the QIAprep spin column by adding 750uL buffer PE. Centrifuge for 1 minute and discard flow through.
8. Place the QIAprep column in a clean 1.5mL microcentrifuge tube. Add 50uL nuclease free water to the center of the QIAprep spin column. Let it stand for 1 minute. Centrifuge for 1 minute.
