實驗室手扎

(1) Mouse embryonic stem cell....medium preparation

(2) Pick-up stable targeting ES cell clone

21th April 2007

Day1.

• After 5~7 days of culture with medium containing G418 and ganciclovir , ES cell showed surviving clones. The following protocol describes critical steps in picking up targeting ES clones and preparing genomic DNA from each clone.

Day 6.

• Drawing line with marker pen in the bottom of each 10-cm culture dish.

• Mark the position of each surviving clone to save the time in searching clones in the next day.

• Calculate the total number of clones in order to prepare the appropriate amount of MEFs growing in 12-well culture dish. Make MEFs ready to use!

Day 7.

• Change Fresh ES medium 1 hour before picking up clones.

• Change ES medium into 5 ml of PBS since serum in ES medium will interfere the proper function of trypsin.

• Prepare 10 ul of trypsin per well in 96-well dish. For beginner without experience, 12 clones/well a time is recommended.

• Place the culture dish with lid open under microscope. Use pipette p20 with filter tip to suck the single ES cell clone. Place the ES cell into one well containing 10 ul of trypsin.

• Place the 96-well into 370c incubator for 2 minutes. At the same time, change MEF medium into ES medium in the 12-well MEFs.

• Add 20 ul PBS into each well and use multi 12 channel pipette loading with 100 ul ES medium to pipette the 12-well trypsin-treated ES clones for at least 30 times in order to resuspand single ES cell .

• Put the 12 channel pipette apart, and recycle each yellow tip (do not mix) with p200 pipette to place single ES clone cell into one 12-well MEFs pre-seeding dish. Label clone number on the lid of each well used.

• Change ES medium without G418 and ganciclovir everyday.

Day 10

• Check everyday to see if the confluence of each well reach 50%. Freeze ES clones at this time.

• When ES cells grow up to 50% confluence, suck ES medium out. Wash ES cell with 1 ml/well PBS, and then add 100 ul trypsin and 100 ul PBS into a well. Place the 12 well dish in 370C incubator for 5 minutes.

• Calculate the number of ES cell clones to be frozen and label each freezing tube including date, knockout gene name, experimenter, and ES cell generation.

• Prepare 20% DMSO in ES medium using 50 ml tube (0.5 ml/ tube).

• Pipetting with filter tip for at least 30 times to resuspand single ES cell.

• Add 1 ml ES cell medium into each well to stop trypsin activity. Pre-load 500 ul 20% DMSO in each tube and then take 550 ul of ES cell resuspension into it.

• Cap the tube with fire-burned forceps, and place the tube into cryol-0C freezing container (Nalgene TM) pre-load with room temperature isopropanol in the bottom.

• Place the Cryol-0C freezing container into -800C refrigerator.

• Seeding the left 650 ul ES cell into the same 12 well dish and add extra-500 ul ES medium into each well. Change medium using MEF medium to culture ES cell for genomic DNA extraction.

• Extract ES cells’ genomic DNA when the ES cell reach 80~90% confluence with standard protocol.