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Need 1.5-2 x 107 cells from a 2 day culture.
1. Cells are harvested as normal, washed x 1 in PBS then taken up at conc. of 1.2 x 10 7 cells/ml in cold PBS.
2. Take x2 Biorad curvettes
A. 0.9 ml (107 cells)
B. 0.167 ml cells (2 x 106 cells ) + 0.73 ml PBS
To A add 15 ug linearised plasmid DNA, IMMEDIATELY prior to electroporation..
B no DNA
3. Using a Biorad Gene Pulser, (McMahon, A.P., and Bradley, A. (1990) Cell 62 1073-1085).
"Zap" each curvette twice i.e. 500 uF 230V then 500 uF 240V
4. ASAP using a pasteur pipette transfer cells from curvette
A to a 10 ml tube containing 4 mls medium, then plate 5 x 1 ml cells onto five 10cm dia petri dishes containing NeoPMEF's and 10 ml medium. Label plates 4-8.
B to a 10 ml tube containing 1ml medium, then plate all onto a 10cm dia petri dish containing NeoPMEFs and 10ml medium. Label plate 2.
5. Set up following control plates.
a) Take 0.167 ml (2 x 106 cells) original cell suspension _ 10cm dia petri dish containing NeoPMEF's and 10ml medium. Label plate 3.
b) Take 0.167 ml (2 x 106 cells) into 10 ml medium (~2 x 105 cells/ml). Plate 0.1 ml (2 x 104 cells) onto Neo PMEF's in 10 cm petri dish with 10 mls medium. Label plate 1.
6. All plates incubated in CO2 incubator overnight.
7. Next day approx. 24 hrs after electroporation plates receive appropriate selection medium.
|Plates||No of Cells||EP||Media|
|1||2 x 104||No EP||Normal||Counting & plating efficiency|
|2||2 x 106||EP No DNA||Normal||% survival after EP|
|3||2 x 106||No EP||G418||% G418 resistant clones in stock|
|4||2 x 106||EP + DNA||Normal||% survival after EP + DNA|
|5||2 x 106||EP + DNA||G418||% total integrants ie random + H/R's|
|6||2 x 106||EP + DNA||G418*||Same as Plate 5 unless|
|7||2 x 106||EP + DNA||G418*||Gancyclovir or FIAU was used -|
|8||2 x 106||EP + DNA||G418*||if so then in theory all should be H/R events (this is not the case!)|
*NB if plasmid contains a TK promoter and positive, negative selection (PNS) is used these plates also receive Gancyclovir or FIAU as well as G418.
Medium is changed on all plates every 48 hrs. By 3 days non G418 resistant clones should be dying off, after one week there should be no clones on Plates 2 & 3. If there are clones present the experiment is invalid. After 6-7 days the colonies on plates 5-8 (plates 6-8 if PNS is used) are ready for harvesting. For efficient picking of single clones there should be no more than approx. 100 colonies/plate.
Picking of single colonies
Plates are treated one at a time and as quickly as possible. Remove medium wash with PBS. Don't wash with PBS/EGTA as this causes the feeders to lift off and the ES cell colonies to float away and break up.
Add a small volume of PBS to plate, hold plate over a piece of black cardboard, using a P-200 Gilson pick colony bringing with it the smallest possible volume of PBS. Pick 6 colonies in succession into a 96 well plate containing 20ul TRYPSIN/EDTA. Check using microscope that there are single colonies in each well. Using a multichannel pipette colonies are dispersed by pipetting up and down 40x in a circular motion being careful not to make bubbles as these cause cell death, check that colonies are dispersed. Cells are then transferred to a 96 well tray, containing Neo PMEF's. This process is repeated until all clonies are picked. After 3-4 days clones should be ready for splitting and freezing down.
Splitting and Freezing down of clones
Treating one row at a time (there may be clones that are not ready), remove medium using aspirator and sterile pasteur pipette, wash each well with 0.1 ml PBS, aspirate, wash with 0.1 ml PBS/EGTA, aspirate, add 40 ul TRYPSIN/EDTA, using a multichannel pipette disperse cells as before, transfer 25 ul into 96 well plate containing 10% DMSO in medium. Then using a Gilson and a sterile tip transfer remaining cells to a 24 well plate with 1 ml medium with LIF (no feeders) in each well, 24 wells are pre-treated with 0.5ml gelatin/PBS. These cultures are for DNA isolation for Southern analysis.
Once entire tray is complete, the 96 well tray is taped up around the edge, occupied wells are recorded on record sheet and tray is then put into a pre-chilled foam box at -70ĄC. Once cultures are frozen > 2 hrs the trays are transferred to an esky at -80ĄC and stored until Southern analysis is complete.
Isolation of DNA from 24 Well plates
Once cultures are almost cofluent, (it doesn't matter if the colonies are very large or if some of the cells have differentiated, as this won't effect the DNA), they are ready for harvesting.
1. Remove medium using aspirator and pasteur pipette.
2. Wash each well x2 with 0.5 ml PBS.
3. Add 0.2 ml 6M Guanidine HCl/NaAc solution, cells lyse almost immediately.
4. Using a pasteur pipette and a rubber teat, slowly suck up the viscous solution. It is important to do this slowly otherwise the DNA will shear. Transfer the solution into a labelled eppendorf tube. The tubes are then taped into an eppendorf rack and taped to the wheel to mix overnight.
5. Next day, in lots of 12 or 20 depending on size of rack used, add 500 ul Abs EtOH to each tube using a pipette aid and a 5 ml pipette. Mix each tube by inversion (don't vortex) until DNA precipitates.
6. Prepare a series of tubes containing 250 ul 70% EtOH, this can be dispensed using multipette eppendorf, and a labelled series with 200 ul TE in each, also dispensed using the multipette eppendorf.
Using a glass capillary tube and holding your forefinger over the end to avoid transferring EtOH along with the DNA, scoop out the DNA, still holding finger over the end of the tube dip DNA into 70% EtOH, wriggle around to wash the DNA then transfer DNA into tube containing TE. DNA should come off the end just by wriggling the tubing, if not close the lid onto the glass and snap off the excess, leaving the DNA attached to the glass.
NB. The transfer of the DNA between tubes must be done quickly, especially the step between the Abs EtOH and the 70%EtOH as the DNA dries out very quickly and is then virtually impossible to redissolve.
7. To dissolve the DNA, tubes are placed in racks taped to the bottom of the shaker incubator in the 32P lab and shaken overnight on speed 10 @ 50ĄC. The solution should be slightly viscous, there may be some undissovled debris in the tube, this will break up by pipetting it up and down. It does not interfere with the restriction enzyme digestion.
8. 75 ul of digested DNA is enough for Southern analysis. This leaves enough for a repeat Southern if necessary.
G418 (Geneticin, GIBCO-BRL 11811-031) need 150ug/ml active (however, each batch should be tested. Make up X100 in PBS and filter sterilize.
NB. Each batch has number of ugs/mg active written on vial, you need to allow for this when making your calculation.
FIAU (1-[2-deoxy, 2 fluoro-§-D-arabino furanosyl]-5 iodouracil)
In eppendorfs in -80ĄC (Made up as 2000X)
Working concentration = 0.2 uM.
Gancyclovir. Freeze dried powder form Syntex. $57.71/vial RMH pharmacy.
6M Guanidine - HCl, 0.1M NaAc (pH 5.5)
add 1/30 3M NaAc pH 5.5
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