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XL Blue Competent Cell Preparation

Materials
12 mL of 25 mg/mL Final Amount to add
Chloramphenicol Concentration
Chloramphenicol 25 mg/Ml Around .3 g
100% Ethanol Fill to reach final concentration (about 12
ml total volume but adjust for actual
amount of chloramphenicol added)
Amount needed: 200 µL

9 mL of Competent Cell Salts Final Amount to add


Solution Concentration
MgCl2 .44 M 4mL of 1M MgCl2
MgSO4 .44 M 4mL of 1M MgSO4
KCl .11 M 1mL of 1M KCl
Instructions: After mixing, filter through a 2 µm disposable filter (syringe filter)
Amount needed: 4.48 mL

500 mL of Transformation Final Amount to add


Buffer Concentration
PIPES 10 mM 1.51 g PIPES
KCl 250 mM 9.32 g KCl
CaCl2 15 mM 1.1g CaCl2ˑ2H2O
KOH To pH 6.7
MnCl2 55 mM 5.44g MnCl2ˑ4H2O
MQ water Fill to reach final volume
Instructions: Mix the PIPES, KCl, CaCl2 and fill to 450 mL with MQ water. Adjust the pH to 6.7 with KOH.
Add the MnCl2. Adjust the volume to 500mL with MQ water.
Amount needed: 102 mL

DMSO
Amount needed: 600 µL

Method

Day 1
Sterilize the bench
Add 1 mL of media to a microcentrifuge tube.
Use a loop to add cells from the competent cell stock to the media.
Shake at 37°C and 210 rpm for 1 hour.
Take 100 µL of the culture and plate the cells on 2xty agar petri dishes with chloramphenicol (cover half
and then streak the other half).
Put them in the incubator with the cap askew for 20 minutes so the liquid evaporates.
Flip them upside down and let them incubate overnight at 37°C.

Day 2
Add 2.24 mL of competent cell salts solution to 100 mL of 2xty in a flask (do this for 2 flasks).
Add 100 uL of chloramphenicol to each media flask.
Innoculate 20 colonies from the plate.
Shake one flask at room temperature overnight at 220 rpm and the other at 18°C using Dr. Liu’s shaker.
Get liquid nitrogen overnight.

Day 3
Set the centrifuge to 4°C and install the swing bucket rotor.
Add 50 mL each of transformation buffer to 2 new falcon tubes. Centrifuge and exchange to fresh falcon
tubes. Store on ice.
When the A600 of the culture has reached .6 (do not use anything over .7, harvest only one of the flasks),
chill the culture on ice for 10 minutes
Pellet the culture by spinning at 4000rpm at 4°C for 10 minutes in 50 mL falcon tubes.
Discard the supernatant (into a beaker with bleach) and drain the excess by letting the falcon tubes
drain on a paper towel.
Resuspend the pellet with 30 mL of ice cold transformation buffer per falcon tube using a 10 mL plastic
pipette.
Incubate on ice for 10 minutes.
Spin at 4000rpm at 4°C for 10 minutes.
Discard and drain excess supernatant as before.
Resuspend the pellet with 4 mL per falcon tube of ice cold transformation buffer
Add 300 µL per tube of 100% DMSO while gently swirling the cell solution.
Incubate on ice for 10 minutes.
Pipette 200 µL aliquots of (200, 400, 800) of cells into sterile 1.5 mL microcentrifuge tubes (pre-chilled).
Store on ice.
Flash freeze the cells with liquid nitrogen and store at -80°C.