MAGE
PROTOCOL DUMP PLEASE FORMAT
Make a stock of MAGE oligos with total concentration of 1-2uM in water. Using stock with concentration of 10uM would doubles the time for each MAGE cycle.
Grow a MAGE strain (EcNR1 or EcNR2) in a culture tube for overnight in 30°C shaker with appropriate antibiotic (in LB miller or LB lennox). EcNR1 has ampicillin resistance. EcNR2 strain has CmR and AmpR.
Measure the overnight OD and dilute the culture to OD=0.05 in 5 ml LB lennox or SOC.
Wait 2-3 hr for the cells to read the OD≈0.5-0.7. The cells grow slightly faster in SOC. (**Turn on the water bath, and also pre-chilled microcentrifuge tube and DW in fridge).
Put the culture tube in shaking water bath at 42°C for 15 min and no more than 20 min.
Add 5 ml media (LB lennox or SOC) to a culture tube and put it in shaker (37°C) to warm up for at least 30 min. This tube will be used at step 12.
Take 1 ml from the culture tube, transfer it to a microcentrifuge tube, centrifuge it at 15,000 RPM for 30 to 60 seconds, and dump the supernatant.
Take another 1 ml of the culture, transfer it to the same microcentrifuge tube, centrifuge for 30 to 60 seconds, and dump the supernatant (doubling cell concentration).
Wash the sample with pre-chilled water (add 1 ml water to the tube, centrifuge, and dump the water)
Repeat step 9 once if the growth media is LB lennox and repeat step 9 twice if the media is SOC.
Add 50 ul of the MAGE oligos to the microcentrifuge tube, resuspending the cells and transfer them to an electroporation cuvette.
Incubate on ice 1 min and then put the cuvette into electroporator (Eppendorf 2510), set the voltage to 2500 and electroporate.
Transfer all the cells from cuvette to prewarmed media tube from step 6.
Put the tube into 30°C shaker to let the cells grow. This is the end of one cycle. It usually take 2-3 hr for the cells to reach the OD≈0.5-0.7 again.
To perform more MAGE cycles, use the culture tube from step 13 and go to step 4.
Comments:
a) To have a good efficiency, everything should be kept cold during washing (step 7 to step 11). b) The step 8 is tricky. Do not try to remove the media/water completely. Only dump the liquid by inverting the microcentrifuge tube for a few seconds. Then use the red pipet to get rid of the rest of media/water. Any try to get rid of all the media in the first place would result in losing most cells and doubling the time of the next cycle.
Chiam Yu: Sometimes, cells will be resuspended in the remaining liquid after you invert the microcentrifuge tube. Use 1 ml-tip to first remove about 900 ul of the supernatant, very slowly. Then, use a 20 ul tip to remove the rest of the supernatant carefully, without resuspending the pellet.
c) After running the last MAGE of the day, wait 1 hr, then add the appropriate antibiotic to the culture tube and put it back to the 30°C shaker. d) SOC is my first choice (Iman) e) If possible, use 32°C, 300rpm instead of 30°C. (Chiam Yu) f) For co-selection MAGE, oligo for restoring or deleting selection marker should be added to 1~2% of the total concentration of oligo mixture.
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