1. Trypsinize cells and count. Wash twice with sterile PBS.
2. Lyse 1.25x107 cells (about one 10 cm dish) in 0.5 ml lysis buffer in an eppendorf. Note that this lysis buffer contains SDS; therefore DNA will have to be phenol/chloroform extracted before PCR or restriction digest.
Lysis Buffer
100 mM NaCl
10 mM Tris pH 8.0
25 mM EDTA pH8.0
0.5% SDS
0.1 mg/ml Proteinase K (add fresh from frozen stock)
3. Incubate the lysate overnight at 50 degrees C. Next day the lysate should be clear and homogeneous.
4. Phenol/chloroform extract the lysate - once with phenol/chloroform, once with chloroform. It will be difficult to remove the aqueous phase with the pipet. Be gentle so as not to shear the DNA.
5. Precipitate the DNA with one volume of isopropanol for 10-15 minutes at room temp. Mix well; you will easily see the DNA.
6. Spin in microfuge at top speed for 20 minutes. The pellet will be clear and snotty and stuck to the side of the tube. Dump out the supernatant, wash with 70% EtOH and spin again. This time the pellet will be white and may not stick to the tube. Pipet supernatant out carefully so as not to dislodge the pellet.
7. Allow pellet to air dry. Do not vacuum dry or you won't be able to resuspend it.
8. Add 100 ul water or TE and let the tube sit at RT awhile. If pellet refuses to dissolve, heat to 50 degrees, or add more liquid.
9. This is good for PCR or digest or Southern.
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Whole Mount Embryoid Body In situ Hybridization using dig-labeled RNA probes
Fixation of Embryoid Bodies (EBs)
1. Transfer dish of Ebs to a 15 ml falcon tube.
2. Wash twice with PBS at room temp. (2x 5 ml washes)
3. Fix in 10 ml of 3.7% formaldehyde in PBS at room temp for
1-2 hours or overnight at 4 degrees.
4. Dehydrate EBs into 100% methanol.
5. Distribute EBs into eppendorf tubes and store at -20 C.
Day 1
Pretreatment of EBs. All washes or treatments were done by adding 1 ml to an eppendorf tube.
1. Rehydrate EBs in methanol/PBT series (3:1, 1:1, 1:3 methanol/PBT).
Finish with 3 washes of PBT. Take about 3 min for each wash.
2. Treat EBs with proteinase K (10 ug/ml in PBT for 10-20 minutes
at room temp).
3. Stop proteinase K treatment by replacing the solution with
3.7% formaldehyde in PBS. Fix for 20 min at room temp.
4. Wash 2x for 5 min in PBT.
5. Ad 1 ml of sHybe and insubate at Room temp for 5 min.
6. Replace with 1 ml of fresh sHybe and incubate at 60-70 C for
at least 2 hours, up to 7 hours.
7. Once in sHybe, EBs can be stored at -20 C. for months.
Hybridization
1. Take digoxigenin-labeled RNA probes out of -80 C. storage and
thaw them on ice.
2. Add desired amount of probe to 200 ul of sHybe in 0.5 ml eppendorf
tubes. The final volume of your hybridization mix should be about
400 ul.
3. Add desired amount of sHybe to EBs so you can later evenly
distribute them into tubes containing probes (you have to do a little math
here)
4. Heat treat probe for 5 minutes at 80 C.
5. Add 200 ul of EBs to each tube containing probe.
6. Mix by inverting a few times and incubate at 60-70 C. overnight.
Submerged in a water bath is good.
Day 2
Posthybridization washes
1. Rinse 2x at hyridization temp in prewarmed hybridization mix.
2. Wash 2x for 30 min at hybridization temp in prewarmed hybridization
mix.
3. Wash for 20 min at hybridization temp in a 1:1 mix of sHybe
and maleic acid buffer (MABT)
4. Rinse 3x in MABT at room temp.
5. Wash 2x for 30 min in MABT at room temp.
Antibody incubation
1. Replace MABT with MABT + 2% Boehringer Blocking powder (MABTB).
2. Wash at room temp for 1 hour.
3. Replace with MABTB + 20% sheep serum (heat treated at 56 C
for 30 minutes and stored at -20 C in aliquots) and incubate for a minimum
of 2 hours at room temp.
4. Replace with fresh solution containing 1:2000 dilution of
alkaline phosphatase-conjugated anti-dig Fab fragments (from BMB).
Incubate overnight at 4 C.
Day 3
1. Rinse 3x with MABT
2. Wash 3x for 60 min each in MABT, shaking or rocking gently
at room temp.
3. Wash at least 2x in NTMT, 15 min each wash. This solution
contains levamisole which blocks endogenous alkaline phosphatase activity.
4. Incubate in NBT/BCIP solution. Mix by inverting.
5. When color has developed (minutes to hours, even overnight
at 4 C) wash 3x with PBT.
6. Fix in 3.7% formaldehyde in PBS overnight to preserve stain.
| Solutions
10x PBS (1 liter)
PBT
CHAPS
Heparin
20X SSC
Yeast RNA
|
sHybe (40 ml)
20 ml 100% deionized formamide 2.6 ml 20X ssc (pH 5.0) 400 ul 0.5M EDTA 2 ml 10% CHAPS 40 ul 50 mg/ml yeast RNA 40 ul 100 mg/ml Heparin pH to 5.0 with 1 M Citric Acid Bring to 40 ml with sterile water Proteinase K
MABT
MABTB
NTMT
|
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RT-PCR Protocol
Noah Byrd, 2000
Necessary items: sterile H2O, 10X PCR buffer (Sigma), taq (Sigma),
dNTP, primers of choice, cDNA, bucket of ice
30.5 ul H2O
5 ul PCR buffer 10X
5 ul dNTP
2 ul 3’ primer
2 ul 5’ primer
0.5 ul taq
45 ul per tube
+ 5 ul cDNA (5ug) = 50 ul total reaction volume
When making the master mix, make enough for one more reaction than you are going to run (this ensures that you don’t run out by the time you get to your last tube).
IMPORTANT:
* master mix: pipet the H2O, PCR buffer, dNTP, 3’ & 5’ primers
and place on ice (add the taq just before you aliquot the mix to your PCR
tubes).
* add 5ul cDNA (well mixed) to PCR tubes at room temperature.
* add taq to the master mix and mix vigorously with pipet set at large volume.
* aliquot 45 ul to each PCR tube, mixing the master mix well and placing on ice between each aliquot, mix, and place on ice.
* keep PCR tubes on ice until ready to place in PCR machine (I always wait until the temp of the heating block has reached 85o).
Don’t be afraid to mix too much, in fact, I encourage it. Get silly!
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In situ hybridization to paraffin sections using dig-labeled RNA probes
Pretreatment of sections:
Materials:
Xylene
Ethanol series
Sterile PBS (CMF)
Sterile 0.85% Saline
Fresh 3.7% formaldehyde
Sterile 50 mM Tris 8.0, 5 mM EDTA (T50 E5)
Acetic Anhydride (toxic, store and use in hood)
Coplin jars (35 ml vol) (C) and glass staining trays (250 ml vol) (G)
1. Dewax slides in xylene, twice for 10 min (G)
2. Remove xylene with 100% EtOH for 2 min (G)
3. Rehydrate by quickly putting slides through 100%, 95%, 85%,
70%, 50%, then 30% EtOH (G)
4. Remove ethanol in salive, then PBS for 5 min each (G)
5. Post fix in 3.7% formaldehyde in PBS for 20 min. (G)
6. Wash with PBS twice for 5 min (G)
7. Proteinase K - 20 ul/ml in T50E5 7.5
min (C)
8. Drain excess and wash with PBS for 5 min (G)
9. Refix in 3.7% formaldehyde in PBS for 5 min (G)
10. To 250 ml 0.1 M triethanolamine, add 0.625 ml acetic anhydride.
Mix well on stirrer.
11. Dip slides in water, then immerse in acetic anhydride for
10 min.
12. Wash with PBS, then saline, for 5 min each (G)
13. Dehydrate by qyuickly putting slides through 30%, 50%, 70%,
85%, 95%, 100% EtOH.
14. Air dry and use on same say for hybridization.
Hybridization and Washing
Materials:
Hybridization mix: pick one according to how strong your probe is: Dextran sulfate buffer gives stronger signal but higher background; buffer B gives cleaner, weaker signal.
Dextran sulfate buffer: 50% deionized formamide; 0.3 M NaCl; 20 mM Tris pH 8.0; 5 mM EDTA, 10 mM NaPO4; pH 8.0; 1x Denhardt's; 0.5 mg/ml yeast RNA, 10% Dextran sulphate (add last) Store in aliquots at -70 C.
Buffer B
0.5 M NaCl, 10 mM Tris pH 7.5, 5 mM EDTA
2 mg/ml RNase A (BMB, prepared as in Maniatis)
20X SSC
Hybrislips (plastic coverslips)
55 C. oven, tupperware box to contain slides on racks
dig-labeled RNA probe (we generally make them according to protocols
from the RNA polymerase manufacturer)
1. Dilute probe as desired. We use dilutions from 1/100 to 1/400 depending on gene of interest.
2. Denature probe at 80 C. for 2 min, then keep on ice.
3. Put about 100 ul of probe onto each slide, then cover carefully with hybrislip. Try not to introduce bubbles during pipetting, as they are very hard to get rid of. Arrange slides on plastic racks in 150 mm culture dishes, with the rack sitting on whatman filters soaked in 5X SSC/50% formamide (for this you don't have to deionize it). Then pack the dishes into a large tupperware container which also is lined with soaked paper towels. Seal the tupperware and put in 50-55 C. oven overnight.
4. In the morning, start preheating the stringency wash solution!
5. Remove the slides and place vertically in a 250 ml staining jar in 5X SSC at 50 C. for 5 minutes for the hybrislips to fall off.
6. Then move them to copelin jars (you can fit eight if you put the middle three back to back) and incubate 30 min at 65 C. in 50% deionized formamide, 2X SSC.
7. Still in copelin jar, wash with 0.5 M NaCl, 10 mM Tris pH 7.5, 5 mM EDTA, once at rt and 2 times for 10 min each at 37 C.
8. Treat with 20 ul/ml RNase A in this buffer for 30 min at 37 C. (You may need to titrate RNase A stock for best signal-to-noise ratio.)
9. Wash with same buffer at 37 C. for 15 min.
10. Repeat step 6, then wash with 2X SSC, then 0.1X SSC for 15 min each.
Antibody Treatment and staining
Materials:
Antidigoxigenin-AP Fab Fragments (BMB)
Buffer 1 (100 mM Tris, pH 7.5, 150 mM NaCl
Buffer 3 (100 mM Tris, pH 9.5, 100 mM NaCl, 50 mM MgCl2
Buffer 4 T10E1 (pH 8.0)
1. Still in copelin jars, incubate in Buffer 1 15 min
2. Dry back of slides