Cre Transfection (noTK selection)
DAY 1. Plate 1 vial of clone onto 1 well of a 24 well (24w) gelatinized plate with feeders
(plate feeders 1 day before) in G148 (or whichever appropriate drug) ES media.
DAY 3. Split to 1 well of a 6w with feeders and G418.
Day 5. Trypsinize and count cells. Need 0.5 - 1 X 107 cells for electroporation. If enough
cells, proceed with electroporation (ZAP). If not enough, plate on 1 10 cm plate with
feeders and G418 and wait two days to ZAP.
Electroporation:
HeBS Buffer:
1M Hepes
5M NaCL
1M KCl
1M Na2HPO4
H2O
2.5 ml
2.68 ml
0.5 ml
70 ul
94.25 ml
a. Wash cells 2X with HeBS
b. Resuspend in 0.75 ml HeBS. Add 50 ug circular Cre DNA (maximum volume
70 ul)
c. ZAP in 0.4 cm gap cuvette
450V
25 uF capacitance
Time constant 0.4 -0.6
d. Plate cells on 1 10cm plate with feeders (NO DRUG)
Day 6.Feed.
Day 7 Trypsinize and count. Plate 200 cells each on 4 10 cm plates with feeders/ no drug.
Day 14 THIS IS USUALLY PICKING DAY
AM Hold plate up to light and circle colonies with marker on bottom of dish.
Look at circled colonies through microscope. Cross out bad looking
colonies (dying or differentiated/fried-egg looking). Idenitfy healthy colonies that have
smooth rounded edges. Try to find colonies that are well spaced from other colonies to
decrease the chance of picking more than 1 colony at a time.
Circle the 96 best colonies over all 5 plates for picking.
PM For picking, you will need:to have ready 4 48 well plates with feeders (Each
plate should have 1 ml of media in each well. In every other row of wells add G418
media), In 96 well plates add 150 ul 0.25% trypsin to every other well in every other
row. Place sterile stuffed P200 tips in every other hole in boxes (ie you'll have 6 tips/
row that will fit every other slot on 12 channel pipetor) You will also need sterile stuffed
p200 tips for picking colonies.
Trypsinize one plate of clones for picking:
Aspirate supernatant
Wash plate with 5 ml 0.05% trypsin
Wash plate rapidly with 2.5 ml 0.25% trypsin. When you aspirate this trypsin,
though, leave behind @ 200-300 ul behind in the plate.
Incubate 70-80 seconde at 37oC, 5%CO2.
(Note: You need to get a feel for how much trypsin to leave on the plate and how many
colonies you can get off a plate quickly. There is a fine balance between too much trypsin
which results in the colonies moving around on the plate and out of your circles, and too
little which results in the plate drying out and the colonies dying before you get to pick
all of the circled ones. Do a 100 plate first to see how it goes.)
Pick clones:
(We do this in the hood on a tower of pipet boxes culminating with an upside
down 10 cm tc plate bottom. Put the plate that you are picking from on top of the upside
down plate. The upside down plate bottom allows your plate to be lit from underneath
which lets you see the colonies better. Having this on a tower brings the whole thing
closer to your eyes so those of us with less than eagle eyesight can see the colonies.)
With P200 pipet preset at 50ul, draw up @10 ul from first well of 96-well plate.
Moving to clone dish, drop trypsin directly onto clone. Hold tip close to clone and suck
up immediately into tip. N.B. YOU WILL BE ABLE TO TELL IF YOU'VE
SUCCESSFULLY PICKED A CLONE BY NOTING IT'S ABSENCE ON THE DISH,
RATHER THAN BY ACTUALLY SEEING IT ENTER THE PIPET TIP. If a clone is
particularly stubborn about coming off the plate, you can sometimes help it along by
scraping or scoring around it with the pipet tip.
Hints: 1. Mark plate with a gird to help keep your place while picking.
2. Keep plate level at all times after addition of trypsin. Any tilt of the
plate after this point will cause the clones to slide. If this happens, any scoring of the
clones that you did prior to picking will be meaningless.
3. Keep plate covered when not actively picking. This will help keep plate
from drying out before you've picked most of the clones.
Transfer picked clone to first well of trypsin plate. Mix vigorously with pipet both to start
breaking up the clone and to create lots of bubbles to help keep your place in the plate.
Transfer picked clones to feeder plate.
After picking all the circled good colonies from 1 plate, use 12-channel pipet
preset at 60 ul with tips in "transfer pattern" (ie. One P200 tip on every other slot, 6 tips
total) to first pipet up and down a few more times one row of picked clones Then
transfer 60 ul of typsin with cells to wells with no G418, and then 60 ul to G418 wells. If
there is still liquid left in 96 well, pick up and transer to no G418 wells. Then take a little
media from G418 wells to wash 96 well well and put back in G418 plate. Repeat picking
steps for next dish of clones to be picked.
DAY15-17 Do not need to feed.
DAY17 Identify which clones will be frozen on Day 11 and which will be trypsinized in
their wells. Prepare labeled and aliquoted cryovials and gelatinized 12-well plates. (see
below)
DAY 18 By this day, some of the clones in the 48-well plates wells that don't have
G418 probably need to be frozen and expanded, while others will benefit from
trypsinizing and replating in the same well. Grow these latter clones 1-3 more days and
freeze and expand as explained below.
Identify which clones are completely dead or partially dead in the G418
rows. Freeze their accompanying no drug wells. Keep plates to follow the G418
wells over the next few days to determine which clones have gone on to die.
Think cold for this procedure…all freezing reagents and vials should be kept on ice at all
times.
Cells should be 40-70% confluent in well. Media color is a quick, easy indicator as to
whether a well is ready to freeze…pale gold color indicates that the cells are ready;
anything more basic than an amber shade means that they aren't dense enough. You get
better results if the cells are not overgrown and are still actively proliferating, so try to
avoid letting the cells grow until the media is "fluorescent yellow." Also, after the first 23 days of freezing/expanding, you will notice that some wells will (a) not look too
healthy or (b) show signs of differentiation. If they're just looking poorly, you can
sometimes rescue them by trypsinizing and transferring them to a new feeder well. If
they're starting to show a little bit of differentiaion, though, it's best to freeze/expand
them immediately, even if the well is only 25-30% confluent.
Prepare 12-well gel-coated plates for DNA splits. Fill each well with 3 ml ES medium.
You'll need 1 well for every clone being frozen. Incubate at 37oC, 5% CO2 until ready to
use.
Dispense 150 ul 2X freezing mixture [20%DMSO/80%FCS] into prelableled freezing
vials (1/clone). Keep on ice. (Use a repeat pipetor.)
Trypsinize first group of clones. Limit the number of clones you trypsinize to less than
18. If you try to do more than 18 at one time, the last clones done will tend to stay in
clumps and won't freeze as well.
Aspirate medium from wells.
Rinse each well with 0.5 ml 0.05% trypsin.
Add ~100 (2 drops from a 1 ml pipette) 0.25 % trypsin to each well. Incubate 5',
37oC, 5% CO2.
Add 150 ul cold FCS (using repeat pipetor) to all trypsinizing wells.
Disperse each clone individually into single cell suspension with P1000 pipette (set on
150 ul). Pipette gently, but thoroughly, 8-10 times until cells are uniformly detached from
bottom of well.
Using same tip, add 150 ul cell suspension to corresponding freezing vial.
GENTLY vortex (setting 3) vial for a second or two to mix cells with freezing mixture.
[Final concentration of freezing mixture is now 10% DMSO and 90% FCS] Place vial
back on ice.
Still using same tip, add remaining cell suspension to corresponding well of DNA
plate. Mix thoroughly to distribute cells evenly in well.
After freezing/expanding 24-36 clones, place vials in foam racks at -80oC overnight
before transferring to liquid nitrogen.
(For clones that you wish to trypsinize in place, add 150 ul ES cell media instead of FCS
to stop trypsinization. Pipet as described above. Add 1 ml ES media and grow 1-3 days
until ready to freeze.)
The plates for making DNA are usually ready about 4-6 days after they were plated.