ELECTROPORATION
1) Feed in the morning, allow to recover for several hours. Approx. 2 confluent 10cm plates will be enough for 4-5 electroporations
2) Trypsinize cells 10 min. or more. Add equal volumes media and pipet vigorously (20-40X). Check in microscope to determine if well dissociated.
3) Centrifuge (setting 3 for 4 min=210g, 500rpm) and resuspend pellet in 10 ml PBS.
4) Do 1:10 dilution (100ul cells, 900ul PBS), add 20 ul to hemocytometer.
5) Count cells in 3-lined area with 25 boxes and calculate
(# of cells / # of boxes) * (25) * (10^4) * 10[=df] * 10[= # of mls] = # of cells/ml
6) Spin down cells and resuspend so that you have 11 * 10^6 cells/ml
7) Add 25ug DNA (25ul, if 1mg/ml) to a 0.5cm cuvette, add 900ul of cells.
8) Zap with 230V, 500 microF at RT on BioRad electroporator. Optimal time constant should be 5-7 microsec.
10) Allow cells to recover in cuvette for 5-10 minutes.
11) Remove cells and snotty dead cells with 1ml pipet into 2ml of media, aliquot onto 4 gelatinized plates filled with 6-7mls of media.
12) Add selection to media after 24 hrs. A typical G418 concentration is 150ug/ml. D. Brown uses 180ug/ml.
11) Feed every day. Pick colonies 9-12 days after electroporation.
PICKING COLONIES
1) Replace old media with 3-5ml PBS
2) Pick colonies with a p20 filter tip racked to 10ul for more control. Suck up 2-3ul of media, push colony, suck up colony with only 2-3ul. Put into round bottom 96-well dishes with 30ul STV.
3) After picking a whole 96-well dish, incubate at 37 degrees for 15 minutes. Add 30ul ES-Media to lysing cells (no need to use selection unless you have a situation where you might get revertants), pipet up and down 20 times. Transfer lysed cells to prewarmed gelatin-treated 96-well flat-bottom plate containing 100ul ES-Media (no selection necessary at this point).
4) Replace media the next day. Wells can hold 200 ul.
5) Replace media 2 times on day 2 since they will be growing quickly.
6) Replace media and split. Keep original plate for freeze down and make DNA plates.
• For example: Remove media, rinse 1x with PBS, add 30ul STV.
• Incubate 15 minutes in incubator. Add 30ul ES-Media.
• Pipet up and down 20X. Leave 30ul in well to freeze down.
• Transfer 15ul to pre-gelatinized, pre-warmed plate holding 100ul media to grow for DNA or PCR.
FREEZING MASTER PLATE
1) Add 25ul 2X Freezing Media (Embryomax, Specialty Media). Pipet up and down gently. (Total volume should now be 30-45 + 25 ul = 55-70ul)
2) Wrap plate in parafilm several times to prevent sublimation.
3) Put in styrofoam box so that the plate slowly freezes in -80.
________________________________________________________________________
ORDER INFORMATION
0.1% Gelatin(500ml) ES-006-B
1X ES-cell freeze media S-002-D
2X ES-cell freeze media ES-0020D
1) Feed in the morning, allow to recover for several hours. Approx. 2 confluent 10cm plates will be enough for 4-5 electroporations
2) Trypsinize cells 10 min. or more. Add equal volumes media and pipet vigorously (20-40X). Check in microscope to determine if well dissociated.
3) Centrifuge (setting 3 for 4 min=210g, 500rpm) and resuspend pellet in 10 ml PBS.
4) Do 1:10 dilution (100ul cells, 900ul PBS), add 20 ul to hemocytometer.
5) Count cells in 3-lined area with 25 boxes and calculate
(# of cells / # of boxes) * (25) * (10^4) * 10[=df] * 10[= # of mls] = # of cells/ml
6) Spin down cells and resuspend so that you have 11 * 10^6 cells/ml
7) Add 25ug DNA (25ul, if 1mg/ml) to a 0.5cm cuvette, add 900ul of cells.
8) Zap with 230V, 500 microF at RT on BioRad electroporator. Optimal time constant should be 5-7 microsec.
10) Allow cells to recover in cuvette for 5-10 minutes.
11) Remove cells and snotty dead cells with 1ml pipet into 2ml of media, aliquot onto 4 gelatinized plates filled with 6-7mls of media.
12) Add selection to media after 24 hrs. A typical G418 concentration is 150ug/ml. D. Brown uses 180ug/ml.
11) Feed every day. Pick colonies 9-12 days after electroporation.
PICKING COLONIES
1) Replace old media with 3-5ml PBS
2) Pick colonies with a p20 filter tip racked to 10ul for more control. Suck up 2-3ul of media, push colony, suck up colony with only 2-3ul. Put into round bottom 96-well dishes with 30ul STV.
3) After picking a whole 96-well dish, incubate at 37 degrees for 15 minutes. Add 30ul ES-Media to lysing cells (no need to use selection unless you have a situation where you might get revertants), pipet up and down 20 times. Transfer lysed cells to prewarmed gelatin-treated 96-well flat-bottom plate containing 100ul ES-Media (no selection necessary at this point).
4) Replace media the next day. Wells can hold 200 ul.
5) Replace media 2 times on day 2 since they will be growing quickly.
6) Replace media and split. Keep original plate for freeze down and make DNA plates.
• For example: Remove media, rinse 1x with PBS, add 30ul STV.
• Incubate 15 minutes in incubator. Add 30ul ES-Media.
• Pipet up and down 20X. Leave 30ul in well to freeze down.
• Transfer 15ul to pre-gelatinized, pre-warmed plate holding 100ul media to grow for DNA or PCR.
FREEZING MASTER PLATE
1) Add 25ul 2X Freezing Media (Embryomax, Specialty Media). Pipet up and down gently. (Total volume should now be 30-45 + 25 ul = 55-70ul)
2) Wrap plate in parafilm several times to prevent sublimation.
3) Put in styrofoam box so that the plate slowly freezes in -80.
________________________________________________________________________
ORDER INFORMATION
0.1% Gelatin(500ml) ES-006-B
1X ES-cell freeze media S-002-D
2X ES-cell freeze media ES-0020D