Wisdom

Be enthusiastic
No sarcasm for at least a year
Explain your logic!
Pick a project that can last at least the first 5 years of your own lab
Seek out your PI
Fight for the best project
You need at least 2 stories, meaning the first one should be done in 2 years, twice the speed of grad school
First year is hard, but you'll get through it
If project doesn't work out, kill it
You want to be a PI. Don't tell anyone otherwise.
You can always call me.

IP protein for mass spec analysis of PTM

Treat cells with
10nM Calyculin A
100uM Sodium orthovanadate
2.5mM Nicotinamide
2.5mM Sodium butyrate
30min 37C

Harvest cells from 5 15cm dishes
Resuspend in cold PBS and transfer to eppies
Spin 1800rpm 5min
Wash again with cold PBS. 4 plates of cells/ tube
Remove PBS and lyse with 500ul protein lysis buffer + 1% protease inhibitor cocktail + phosphatase inhibitors
Lyse on ice with occasional vortex 2h

Prepare Dynabeads (Invitrogen)
Completely resuspend Dynabeads by rotating for 5min or vortex 5s
Transfer 50ul beads to eppie
Separate on magnet until supernatant is clear and remove supernatant
Remove tube from magnet

Bind Antibody
Add antibodies (20-30ul of M2 for each IP) up to 200ul Ab binding and washing buffer
Incubate with beads 1h 4C rotate
Place tube on magnet and remove supernatant
Remove tube from magnet and resuspend beads in 200ul Ab Binding and Washing buffer. Wash by gentle pipetting

Immunoprecipitation
Place tube on magnet and remove supernatant
Add lysate to beads and gently pipette to resuspend the beads (keep 5ul as input to run gel)
Incubate O/N 4C rotate

Wash
Place tube on magnet. Transfer supernatant to clean tube (keep for gel)
Wash beads 4x in 1ml TNMEN-150. Mix on shaker 5min 4C
Keep all wash for gel
Resuspend beads-Ab-Ag complex in 100ul Wash buffer and transfer to clean tube to avoid co-elution of proteins bound to tube wall.

Elution
Place tube on magnet and remove supernatant
Add 20ul elution buffer (50mM glycine pH2.8) and 4ul 6x SDS sample loading buffer. Pipette to resuspend
Boil 5min
Store all washes, superatant, IP products at -20C

Run gel
2ul Input, 22ul IP for Coomassie stain
2ul Input, 2ul IP for Western

To stain Coomassie
Rinse peptide gel in ddH2O
Fix gel in 40% Methanol, 10% Acetic aci 30min
Add 50ml Coomassie safe or enough to cover gel. Shake 20min-1h
Wash in ddH2O 2h. Bands will appear during wash
Store in water

To prepare for mass spec
Cut band from Coomassie stained gel with fresh blade
Store in clean tue in 1% acetic acid

Protein lysis buffer
400mM NaCl
10mM HEPES
0.1mM EGTA
0.5mM DTT
5% glycerol
0.5mM PMSF
1% TritonX-100

Phosphatase inhibitors
1% Sigma phosphatase inhibitor 2
1% Sigma phosphatase inhibitor 3
5mM sodium butyrate

TNMEN-150
50mM Tris-HCl pH8
0.5% NP40 (Igepal)
1mM EDTA
2mM MgCl2
150mM NaCl
Add protease inhibitor and phosphatase inhibitors before use

Whole mount in situ hybridization

Fix embryos O/N 4% PFA/DEPC PBS
Wash 2x 5min DEPC PBS
Dehydrate through 50% MeOH/ PBS DEPC, then 2 changes of 100% MeOH
Store -20C
Day 1 (DEPC all solutions): Pretreatment and Hybridization
From dehydrated embryos in MeOH, rehydrate through 75%, 50%, 25% MeOH/ PBST. Just let embryos sink, don't need to rock
Wash PBST 2x 5min
Treat with 5ug/ml Proteinase K in PBST 5-20min depending on size (for chick ~ 1min/ stage)
Remove proteinase, rinse briefly with PBST
Post-fix with 4% PFA + 0.1% Glutaraldehyde/ PBST
Rinse with PBST
Rinse once in 1:1 PBST/ hybridization mix. Let embryos settle
Rinse with 1ml hybridization mix. Let embryos settle
Replace with 1ml hybridization mix and incubate horizontally 1h 65C
Add 1ml pre-warmed hybridization mix with 1ug/ml DIG-labelled RNA probe. Incubate horizontally at 65C. Rotate.
Day 2 (Don't need to DEPC): Post-hybridization washes
Rinse twice with 65C hybridization mix
Wash 4x 30min 1.5ml prewarmed hyb mix 65C
Wash 10min 1.5ml prewarmed 1:1 hyb mix/ MABT 65C
Rinse 2x 1.5ml MABT
Wash 1x 15min 1.5ml MABT RT
Incubate with 1.5ml MABT + 2% Boehringer Blocking Reagent (BBR) RT 1h
Incubate with 1.5ml MABT + 2% BBR + 20% heat-treated sheep serum RT >1h
Incubate with 1ml 1ml MABT + 2% BBR + 20% serum + 1:2000 AP-anti-DIG Ab O/N 4C

Day 3: Washes
Rinse 3 times with 1.5ml MABT
Wash 6-8x 1h with 2ml MABT
Wash O/N 4C

Day 4: Development
Wash 2x 20min with NTMT
Incubate BM Purple AP substrate RT dark
Check after 30min
When ready, rinse with PBST and wash 2x
Postfix in 4% PFA 2h RT
Rinse with PBSt and wash twice
Store 4C

PBST
PBS + 0.1% Tween 20

Hybridization mix50% Formamide
1.3x SSC
5mM EDTA, pH 8
50ug/ml Yeast tRNA
0.2% Tween 20
0.5% CHAPS
100ug/ml Heparin
DEPC H2O

Hybridization mix
Formamide  25ml
20X SSC pH5 with citric acid  3.25ml
0.5M EDTA pH8  0.5ml
20mg/ml Yeast tRNA   125ul
10% Tween 20   1ml
10% CHAPS   2.5ml
50mg/ml Heparin   100ul
H2O   17.5ml
Total  50ml

5x MAB
Maleic acid  11.6g (pH to 7.5 before adding NaCl, neutralize with 10N NaOH, careful after pH 5)
NaCl  8.7g
H2O  185ml
Total  200ml
Dilute with H2O and add 0.1% Tween 20

10% Boehringer Blocking reagent
10% in MAB.
Heat to dissolve. Autoclave. Aliquot and freeze
NTMT (make fresh)
5M NaCl  1ml
2M TrisHCl pH9.5  2.5ml
2M MgCl2   1.25ml
10% Tween-20   5ml
H2O   40.25ml
Total  50ml

Cryo-section In Situ Hybridization

Day 1 (DEPC solutions):
Thaw slides
Post fix in fresh 4% PFA/ DEPC PBS 10min. Save the PFA
Wash 2x 5min DEPC PBS
Incubate 2min in 5ug/ml Proteinase K/ DEPC PBS (Time sensitive)
Wash 5min DEPC PBS
Fix in 4% PFA from before 5min
Wash 5min DEPC PBS
Acetylate sections for 10min with acetic anhydride + 0.1M TEA-HCl pH7.5. Add acetic anhydride to TEA immediately before use.
Wash 5min DEPC PBS
Dehydrate 5min in 70% EtOH, 2min in 95% EtOH
Air dry comepletely
Wash 30-45min in Tris/Glycine Buffer
Prepare hybe mix 150-200ul/ slide
Add 1ug/ml probe
Heat hybe with probe to 80-90C, store on ice
Add 150-200ul hybe with probe onto the slide, cover with a hybrislip
Hybe O/N at 65C. Add damp paper towels to prevent drying

Day 2 (No need to DEPC):
Wash 3x 20min 5x SSC RT
Wash 40min 60C 0.5x SSC 20% formamide
Change wash and place at RT until 37C
Wash in NTE 15min 37C
Wash in NTE + 10ug/ml RNase 30min 37C
Wash in NTE 15min 37C
Wash in 0.5x SSC 20% formamide 30min 60C
Wash 2x SSC 30min RT
Place in 1% blocking solution for 10min at least
Add anti-DIG 1:5000 in blocking solution
Incubate O/N at 4C in humidified chamber

Day 3:
Wash slides 4x 10min, 1x 20min in TBS
Wash slides 10min in 0.1% Tween-20, 0.5mg/ml levamisole hydrochloride in water
Warm BM Purple to RT, mix
Add 10ul of 100x levamisole/ tween-20 per 1ml of BM purple
Place about 170ul of staining solution per slide. Cover with hybri-slip. Incubate RT dark.
Stop reaction by washing slides 10min PBS +1mM EDTA
Dry slides mount with DAKO mounting media.

TEA-HCl pH8
Triethanolamine-HCl  3.71g
DEPC H2O  199ml
10N NaOH  900ul
Total  200ml

Acetic anhydride/ TEA-HCL
Acetic Anhydride  125ul
0.1M TEA-HCl pH7.5  50ml

Tris/Glycine buffer
Tris base  24.2g
Glycine  15g
DEPC H2O to 2L

Hybridization mix
40% Formamide
5x SSC
1x Denhard's
100ug/ml fish testis DNA
100ug/ml yeast tRNA

NTE
0.5M NaCl
10mM Tris pH7
5mM EDTA

Blocking
1% Boehringer blocking reagent in MABT

MABT pH7.5
100mM maleic acid
150mM NaCl
0.1% Tween 20

100x Levamisole/ Tween-20
50mg/ml Levamisole
10% Tween-20

Synthesis of Labelled RNA probe (Roche)

Linearise DNA
Digest 5ug plasmid O/N in 40ul

Transcription mix
Mix the reagents in the following order
DEPC water
10x Transcription buffer  2ul
Nucleotide mix (DIG or FLUORESCEIN)  2ul
Linearised plasmid  1ug
RNase Inhibitor (40U/ul)  1ul
SP6, T7 or T3 polymerase (10U/ul)  1ul
Total  20ul

Incubate 37C 2h
Run 1ul on agarose gel with linearised plasmid to see if probe is synthesized, run fast <15min. An RNA band, 10 fold more intense than the plasmid should be seen.

Precipitation
For DIG-labelled probe:
TE (50mM TrisHCl, 1mM EDTA, pH8.0)  100ul
4M LiCl  10ul
Ethanol  300ul

For FLUORESCEIN-labelled probe:
DEPC H2O  100ul
Isopropanol  300ul
5M NH4 acetate  78ul

Mix and precipitate at -20C 1h-O/N
Spin 10min max 4C
Remove supernatant
Add 70% EtOH
Spin 5min 4C
Wash again with 70% EtOH
Air dry
Resuspend in 50ul DEPC H2O
Spec concentration

Store -80C

10x transcription buffer
40mM Tris HCl pH8.25
60mM MgCl2
20mM Spermidine

Nucleotide mix
10mM GTP
10mM ATP
10mM CTP
6.5mM UTP
3.5 mM DIG or FLUO-UTP pH8

A regulatory ribosome

Isn't it exciting? To think that ribosomes might have specialized activities, that there might be different ribosome populations translating different subsets of mRNAs. I'm suddenly reminded at how exciting this is. How revolutionary this thinking is..

Cloning methods

Strategies for Cloning PCR Products
Peter D’Arpa
Adapted from PCR Primer, 2nd edition (eds. Dieffenbach and Dveksler). CSHL Press, Cold Spring Harbor, NY, USA, 2003.

qPCR

From trypsinized embryo, resuspend in 50ul PBS
Use 15ul and add 500ul Trizol
Store at -80C

RNA extraction
RNaseZAP everything
From cells in Trizol, incubate RT 5min to permit complete dissociation of nucleoprotein complexes
Add 100ul chloroform
Shake vigorously by hand 15s and incubate at RT 2-3min
Spin max speed 15min 4C
Transfer top aqueous phase to fresh eppies (~200ul)
Precipitate RNA from aqueous phase with 250ul isopropanol
Incubate RT for 10min
Spin max speed 10min 4C
Remove supernatant
Wash RNA pellet once with 75% EtOH (DEPC) 750ul
Spin max speed 5min 4C
Remove EtOH, briefly dry RNA pellet
Dissolve in DEPC H2O 20ul. Pipette up and down a few times to completely dissolve
Incubate at 55-60C 10min to dissolve

DNase treatment (25ul)
10x Turbo DNase buffer 2.5
Turbo DNase 1
RNA 20
DEPC H2O 1.5

Incubate 37C 30min
Add 0.1 volume DNase Inactivation Reagent (resuspend before adding) (2.5ul)
Mix well
Incubate 5min RT. Mix 2-3 times
Spin 10000g for 1.5min and transfer RNA to new tube

cDNA synthesis (use PCR machines for incubation)
RNA (up to 5ug) 8
50ng/ul random hexamers 1
10mM dNTP mix 1
Total 10ul

Incubate 65C 5min
Place on ice for at least 1 min

10x RT buffer 2
25mM MgCl2 4
0.1M DTT 2
RNaseOUT (40U/ul) 1
SuperScript III RT (200U/ul) 1
Total 10ul

Add 10ul of cDNA synthesis mix to each RNA/ primer mixture
Mix gently and spin down
10min 25C
50min 50C
5 min 85C
Chill on ice

Add 1ul of RNase H to each tube to remove RNA template
Incubate 20min 37C

QPCR
SYBR Green qPCR Supermix 25
5uM F primer 3.6
5uM R primer 3.6
cDNA 2
DEPC H2O 15.8
Total 50ul

50C 2min
95C 10min
95C 15s
60C 60s
Repeat Steps 3 and 4 39x
Melting curve

B-Gal ELISA

After trypsin and resuspending in media
Spin and resuspend in 50ul PBS
Used 35ul from the 50ul and add 100ul lysis buffer (dilute from 5x B-gal lysis buffer, Soln 8)
Rotate at RT 20min
Store at -80C
Spin max speed 15min 4C to remove cell debris
Use supernatant for ELISA

Make standard
12.5ul of 38ng/ml B-gal enzyme stock solution (Soln 1 -20C) in 500ul of sample buffer (Soln 7, 4C) = 950pg/ml
Serial dilution down to 15pg/ml (250ul + 250ul)
Keep blank

Pipette 200ul of standard dilutions or 200ul of samples into wells (I use as much embryo samples as I can to get good signal), make up to 200ul with sample buffer
Seal with adhesive film, incubate 1h at 37C
Dilute washing buffer from 10x (Soln 6, 4C)

Wash 3x 30s with 1 washing buffer 250ul each
Dilute Anti-B-Gal-DIG solution (Soln 2, -20C) 100x from 50ug/ml to 0,5ug/ml in sample buffer
Pipette 200ul of diluted anti-Bgal-DIG into wells
Seal, incubate 1h 37C

Wash 3x30s
Dilute Anti-DIG-POD (Soln 3, 4C) 133x from 20U/ml to 150mU/ml in sample buffer
Pipette 200ul diluted anti-DIG-POD into wells
Seal, incubate 1h 37C

Wash 3x30s
Add 1mg of substrate enhancer (Bottle 5, 4C) per ml of ABTS substrate solution (Soln4, 4C)
Mix by stirring for 30min RT
Pipette 200ul of POD substrate (Soln 4+5) into wells
Incubate RT 15-40min, shake

Measure absorbance at 405nm with a plate reader. Colour stable for a few hours.

Western from embryos

Remove membranes from embryo
Remove limb buds
Drag off heart + other organs (kidneys, gut) from ventral side of embryo
Cut embryo right above 1st somite
Pin down embryo dorsal up and cut off everything outside somites. Scrape off things beneath embryo too
Store in filming media until ready for trypsinisation
Protein prep
Trypsinise with 1% Trypsin 45min-1h 37C in eppie
Remove trypsin
Resuspend cells with 1ml FM with pipette
Spin 1500rpm 5min
Wash with 1ml PBS
Spin 1500rpm 5min
Add 10ul 10x protease inhibitor into 1ml of protein lysis buffer
Lyse cells with 100ul protein lysis buffer for each embryo 30min on ice
Vortex occasionally
Spin max speed 4C 10min to remove debris
Transfer supernatant to fresh eppies
Store -20C

Bradford assay

Dilute Bradford reagent 1ml in 4ml of water (5x dilution)

Add 1ml of Bradford to each cuvette

Make 5 standards
0.25, 0.5, 1, 2, 4 ug from 1ug/ul BSA
Add 8ul of 1ug/ul BSA to the 4 ug standard
Add additional 1ml of diluted Bradford to 4ug standard
Mix and transfer 1ml to 2ug standard
Mix and transfer 1ml to 1ug etc
Dump the last 1ml
Add 1ul of protein sample into cuvette with 1ml diluted Bradford
Keep a Blank with only Bradford
Spec
Prepare samples for loading by adding 6x Protein loading buffer
Boil (95C) for 5min

Load 20ug
Criterion Tris-Tricine Peptide Gel
Remove tape at bottom, insert into gel tank
Load 12ul of marker
Well volume 30ul
Run 110-125V constant (Start current 140-150mA/gel. End current 60-70mA/ gel)
Run 2h

Running buffer (Dilute from 10X Tris-Tricine-SDS buffer)
100mM Tris
100mM Tricine
0.1% SDS
460ml x2 for 1 tank

Homemade acrylamide gel
1.5mm
20ml resolving for 2 gels
8ml stacking for 2 gels
10 wells can load up to 50ul

Run stacking at 80V
Run resolving at 120V
Running buffer dilute from 10x
Semi-dry transfer
Cut everything to same size
Soak filters and membrane
Assemble sandwich (gel on membrane)
Transfer at 45mA 1h
Put something heavy on transfer apparatus

Transfer buffer (200ml)
Tris-Glycine-SDS (dilute from 10x Tris-Glycine-SDS running buffer)
20% MeOH

Wet transfer
Soak sponge, filter paper and membrane in buffer
Assemble cassette, black at bottom, membrane on gel
Place cassette in tank, black side towards black
Fill tank with buffer
Transfer 4C O/N 17V or 90V on ice 2h with ice pack
Transfer buffer (1L)
Dilute from 10x transfer buffer
20% MeOH

Antibody incubations
Remove membrane and stain with Ponceau
Rinse off with H2O or PBST 3x
Block with 5% milk in PBST 1h
Incubate primary antibody (10ml in box, 1ml in plastic seal)
Wash membrane with PBST 3x 20min
Incubate with secondary antibody 1:1000 RT 1h
Wash PBST 3x 20min
Turn on developer during last wash

Development
Mix SuperECL solutions 1:1 (2ml/ membrane)
Incubate with membrane 5min
Put membrane in cassette
Develop

Immunostaining

Equilibrate slides to RT
Rehydrate in blocking buffer for 45min-1h in Coplin jar
Incubate with primary antibody in blocking buffer 4C O/N in humidified chamber
Use 170ul Ab/slide cover with hybrislips. Use water soaked Kimmies to create humidified chamber

Remove hybridslips
Wash 20min x3 with blocking buffer
Incubate with secondary antibody in blocking buffer 1h RT dark
Wash 20min x3 with blocking buffer
Mount slides using VectorShield + DAPI (1 drop/ slide) with glass coverslip
Seal with nail polish
Dry
Store at 4C dark

Blocking buffer: 1% heat-inactivated goat serum in PBS + 0.1% TritonX-100

Cryosections

Equilibrate fixed and washed embryos in sucrose (30% sucrose in 0.1M potassium phosphate buffer pH7.4) O/N 4C

Mounting
Cut section of embryo for sectioning with sharp blade
Transfer wanted section to molds with cold OCT on ice
Let tissue sink in OCT ~1h on ice
Place some crushed dry ice in tray and put OCT block in tray
Orient embryo such that anterior is towards bottom of block and embryo is standing upright
Transfer OCT block carefully to flat surface of dry ice
Keep blocks at -80C until ready to section

Sectioning
Equilibrate blocks in cryostat to -17C - -18C for at least 30min
Section 10um sections
Store slides at -80C

LysoTracker Red staining for apoptotic cells

LysoTracker is an acidophilic dye. Stains lysosomes. Vital dye to indicate apoptotic cells
Transfer embryos to 5uM LysoTracker Red in Hanks BSS in RNase-free non stick tubes
Incubate at 37C for 30-45min
Rinse 4-5x in Hanks, shake RT 5min each
Fix in 4% PFA/PBS 1h at 4C, shake
Wash with PBST 15min 4C shake
Wash PBS 3x 15min 4C shake

Chick protocols

Chick staging table
Turn eggs so they lie on sides
Incubate at 37C 40-45h to HH11 (3 primary brain vesicles, heart bent to right)

Window eggs
Clean eggs with 70% EtOH
Poke hole on blunt end of egg and small hole on top of egg
Suck out 5ml of albumin from blunt end of egg with syringe to lower embryo from shell
Tape up hole on blunt end
Tape top of egg. Cut a circular window through tape and shell
Cover hole tightly with tape

Pull glass needles
Solenoid: 2
Heating: 15
Follow instructions. Clamp top and bottom such that centre of capillary tube is at solenoid
Close cover and press START
Solenoid heats up and clamp pull and break capillary tube
Store needles in Petri dish

Injection into neural tube
Load as little as 3ul into glass needle (1ug/ul DNA + 1/10 1% Fast Green or 1mM morpholino + a bit of 1ug/ul plasmid DNA)
Break off end of glass needle
Fit needle into PicoSpritzer
Inject into neural tube till entire neural tube is filled
Add filming media + 10x AA to cover embryo (2-4ml)
Place electrodes on either side of embryo
Electroporate (See bubbles), make sure electrodes don't stick to embryo
Seal up window with tape and incubate at 37C

Injection settings
35ms
6 spritzes

Electroporation settings
25V
50ms
3 pulses

Dissection
Check embryo is alive (heart is beating)
Cut around it and pick up with scoop
Transfer embryo to Hanks' BSS, Ca2+, Mg2+ free, w/o Phenol Red
Dissect out embryo by removing extra embryonic membranes

Lab choice advice

"I want to join a lab where I'm a f*cking moron, so I can learn from everyone"
"It always help to have a colleague in the same phase of career as you are, so you can bounce ideas off each other"
"You're here to train to think, it doesn't matter what you work on. You won't work on it in your postdoc or in your own lab anyway"
"Productivity is kind of important as a graduate student. Go to a lab where you're productive" -Josh
"Don't feel like you can only do what (PI) tells you to, do that as a hobby" -Patrick
"If you join you'll see lots of pictures of a crazy French woman and her fat baby" -Nat
"If you have problems with people in the lab, you'll just be miserable day to day. If you have problems with the PI, you might not get your PhD" -Ben

Being in that lab for 30min reminded me of how awesome those people are. And of course Salma took the chance to harass my ass

"Expts often fail the first time. Don't take it on yourself"
"This is not like in situs where u know what to expect. It's real science"
"Join the lab where u're most interested in the science. It's what keeps u going ultimately"

Chick neural tube electroporation

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2566325/

In situ hybridization

From dehydrated E9.5 embryos, rehydrate embryos in 75%, 50%, 25% MeOH/PBT 5min each shaking
Wash 2x PBT 5min RT
Bleach with 6% H2O2 in PBT 1h RT (~6ml each vial)
Wash 3x PBT 5min RT
Permeabilize with 10ug/ml Proteinase K in PBT RT (2.5min for E7.5, 5min for E8, 10min for E9, 20min for E10-11)
Wash with 2mg/ml Glycine in PBT 15min RT to stop ProK reaction
Wash 2x PBT 5min
Postfix with 4% PFA/ 0.2% gluteraldehyde in PBT 20min RT
Wash 2x PBT 5min
Pre-hybridize with warm hybridization buffer 1h
Add 2ml probe to each vial
Hybridize O/N 70C

Remove probes from vials and keep at -20C to reuse 5-6 times
Rinse with warm Wash Solution I
Wash with Wash Solution I 3x 30min 70C shaking
Wash with Wash Solution II 3x 30min 65C
Wash with TBST 3x 5min RT
Preblock with 10% heat-inactivated sheep serum in TBST 1-2.5h
Remove block. Add anti-dig antibody 1:5000 in 1% sheep serum
Rock O/N 4C

Wash embryos in TBST 3x 5min RT
Wash 6-8x 30min-1h TBST RT
Wash O/N TBST 4C

Wash with fresh NTMT 3x 10min
Incubate with RT BM Purple. Cover with foil. RT-37C
Check after 20min
Develop till desired darkness
Rinse briefly in NTMT 2x 5min
Wash with PBT pH5.5 2x 10min RT to stop reaction
Post fix with 4% PFA 1h RT or 4C O/N
Wash 2x PBT
Dehydrate through Methanol row. Keep at -20C

DEPC water
0.1% DEPC in H2O. Sit O/N. Autoclave

PBT
PBS (DEPC) with 0.1% Tween 20. Filter

Hybridization solution (500ml)
Formamide 50% 250ml
20x SSC pH4.5 5x 125ml
50mg/ml yeast tRNA 50ug/ml 500ul
10% SDS 1% 50ml
50mg/ml heparin 50ug/ml 500ul
DEPC H2O 74ml
Filter
Aliquot into 50ml and store at -20C

Wash solution I
Formamide 50%
20X SSC pH 4.5 5x
10% SDS 1%
DEPC H2O

Wash solution 2
Formamide 50%
20X SSC pH4.5 2x
Tween 20 0.1%
DEPC H2O

NTMT (100ml)
5M NaCl 100mM 2ml
1M Tris pH9.5 100mM 10ml
1M MgCl2 50mM 5ml
Tween 20 0.1% 0.1ml
1M Levimasole 2mM 200ul
H2O (non-DEPC)
Filter

Riboprobe preparation for in situ hybridization

Promoter- DNA with same sequence as mRNA needed

Linearize 10ug of plasmid DNA using 10x excess of appropriate enzyme (10U to 1 ug of plasmid DNA) 3h - O/N
Run 1ul on gel to verify complete cutting
Treat with 0.1ug/ul of Proteinase K for 30min at 37C to eliminate RNases and polymerases
Purify with Miniprep column. Elute in 31ul of warm Rnase-free H2O (let stand 1-2min before spinning). Place on ice.
Dilute 1ul of reaction 1:10 with 9ul H2O. Load 1ul and 5ul with 5ul ladder to quantify linear DNA/ul

Use Roche in vitro transcription reagents and assemble the following components in order on ice
1-2 ug of linearized template
10ul Roche 10X DIG UTP mix
10ul Roche 10x transcription buffer
5ul Roche Protector RNase Inhibitor
Make up to 90ul with RNase-free H2O (not DEPC)
Then add 10ul Roche T7 or SP6 or T3 depending on the promoter
Mix, spin down, wrap in parafilm and incubate 37C 2h

Remove tube from water bath, add 10ul Roche Dnase I (10U/ul) and incubate at 37C for 15min to destroy DNA template
Add 4ul 0.5M EDTA to terminate DNase reaction. Keep tubes on ice.

Purify with Roche Quick Spin columns.
Tap off top cap, remove bottom cover and drain fluid
Place column in small colletion tube and place both in 15ml Falcon tube and spin 800g for 2min
Change collection tube to fresh collection tube and load reaction (110ul) to center of column and spin 800g for 4min.
Transfer purified probe to fresh eppies
Add 1/9th volume 3M NaAcetate (RNase-free), pH5.5 (12.2ul for 110ul reaction). Mix well and add 3 vol of ice cold 100% EtOH (RNase-free). Mix well and ppt at -80C 3h-O/N

Spin at 4C max for 30min
A large white pellet should be visible. Remove as much EtOH as possible
Add 700ul cold 75% EtOH (RNase-free) to wash DNA
Spin 4C 15min
Remove as much EtOH as possible, air dry for 1-2min
Add 21ul RNase-free H2O, mix gently, then place tube at 55C with lid open to remove any residual EtOH.
Dilute 1ul probe 1:10 and run 1ul and 5ul and marker on agarose gel. Run fast (150V)

Bring probe in top quality formamide HYB buffer to final concentration of 0.1ug/ul and store -20C for months

B-Galactosidase staining

Keep uterus in cold DMEM/F12 until ready to dissect
From dissected embryos in PBS
Fix embryos in Fixation solution shaking on ice for 5min (E7-9) to 15min for adult hearts
Wash with cold PBST 2x
Permeabilize with Permeabilization solution shake at RT for 20min-1h
Stain with X-Gal Staining solution at RT until desired colour
Post fix with 4% PFA 4C O/N

Make solutions fresh each time
Fixation (5ml)
37% Formaldehyde 2% 270ul
25% Glutaraldehyde 0.2% 40ul
Permeabilization buffer 5ml

Permeabilization (500ml)10% NaDeoxycholate 0.02% 1ml
10% NP40 0.01% 0.5ml
PBS 496.5ml

X-Gal Staining solution (5ml)50mM K-Ferricyanide 5mM 500ul
50mM K-Ferrocyanide 5mM 500ul
200mM MgCl2 2mM 50ul
100mg/ml X-Gal 1mg/ml 50ul
PBS 4ml
Filter through 0.45 or 0.22um filter