Embryo Preparation at 8.5 dpc to 11.5 dpc

  • Mice are killed by cervical dislocation. Uteri are dissected and placed in chilled PBS.

  • Remove all extra-embryonic tissues from the embryos under a binocular.

  • Gently open the brain ventricles with your tweezers tip before transferring the embryos into 4% PFA/PBS. This will help to avoid background staining.

  • Incubate the embryos on a roller at 4°C in 4% PFA/PBS overnight.

  • After this, embryos should be transferred into 100% methanol via a methanol series the next day.


If not stated differently each step includes at least 10 min of incubation while rolling at 4°C.

  • Wash twice with PBS-DEPC

  • Once with 25% methanol/PBS-DEPC

  • Once with 50% methanol/PBS-DEPC

  • Once with 75% methanol/PBS-DEPC

  • Twice with 100% methanol

  • The dissected embryos are stored in 100% methanol at -20°C


Processing of 8.5 dpc to 11.5 dpc Mouse Embryos Before
“Whole Mount” In Situ Hybridization

Dissected embryos are stored in methanol at -20°C. For processing, pool the desired number of embryos of each stage from several dissections. The solution volume for processing steps should be at least ten times the volume of the pooled embryos. Choose an RNase-free container accordingly (e.g. Eppendorf cups, 15 or 50 ml Falcon tubes, 25 ml sterile tubes, DEPC-treated glass bottles). All solutions should be handled with baked Pasteur pipettes, sterile plastic pipettes or by decanting. Exchange the solutions as completely as possible. Wear proper gloves to avoid RNase-contamination.

Formamide contained in Hyb is toxic. Glutaraldehyde and Paraformaldehyde used for fixation are toxic. Handle these solutions with care! Use gloves. Avoid inhalation of vapours! Collect used solutions in special containers.


If not stated differently, each step includes at least 10 min of incubation while rolling at 4°C.

  • Wash once with 75% methanol/PBST-DEPC

  • Once with 50% methanol/PBST-DEPC

  • Once with 25% methanol/PBST-DEPC

  • Twice with PBST-DEPC

    One wash is enough. During the H2O2 treatment remaining methanol does not harm. After this treatment the embryos are washed in PBST-DEPC again.

  • Incubate in 6% H2O2/PBST-DEPC while rolling at 4°C

    According to a revision of the Wilkinson protocol a 1-hour H2O2 treatment reduces the signal strength. However, if H2O2 treatment is skipped completely, the embryos are much less transparent. Therefore, we reduced the incubation time: 10 min for 8.5d, 20 min for 9.5d, 30 min for 10.5d, 45 min for 11.5d and as long as it takes to get white embryos for older stages. Especially the liver should become yellow instead of red.

    H2O2 treatment can be done after staining instead; same result as if done before hybridization

  • Wash 3 times with PBST-DEPC while rolling at 4°C.

  • Incubate at 4°C with 10 µg/ml Prot.K/PBST-DEPC (stock 10 mg/ml) while rolling:


Prot K (Freiburg-Stock)


Prot K (Berlin-Stock)


age (dpc) / Theiler stage

incubation time


incubation time

8.5/12

10 min


7 min


9.5/15

14 min


10 min


10.5/17

16 min


13 min


11.5/19

20 min


17 min

    The incubation times are examples for using Prot.K/PBST-DEPC pre-equilibrated to 4°C and different stocks

  • Wash once with 2 mg/ml Glycin/ PBST-DEPC (stock 200mg/ml) while rolling at 4°C

  • Wash twice with PBST-DEPC while rolling at 4°C

  • Refix for 30 min with cold 0.2% Glut./4% PFA/PBST-DEPC while rolling at RT.

  • Wash twice with PBST-DEPC while rolling at RT

  • Replace PBST-DEPC with Hyb, incubate 15 min at RT while rolling

  • Exchange Hyb and prehybridize 2 h at 68°C

    Afterwards you can proceed immediately with the "whole mount" in situ hybridization protocol or store the prehybridized embryos at -20°C.

Preparation of Probes for “Primary Screen-In Situ Hybridization” Involving In vitro Transcription

This Process must be RNase free! Use clean gloves, sterile solutions and RNA solutions and chemicals.

For longer-term storage of probes after hydrolyzis, re precipitate following steps 7-12 and resuspend pellet in DEPC-water.

    Probes are obtained from the RZPD (www.rzpd.de) in 96 well plates (E.coli cells in LB-medium).

    Defrost the plate gently at 4°C. Using a 12-multi-channel pipette, take up 25 µl of the bacterial culture and release it again.

    Prepare a new 96 well PCR plate with PCR mix wash out the contaminated pipette tips with the PCR mix and run your PCR cycle.

Mix the following PCR mix into a 96 well PCR plate (1 reaction => 50µl):


Example for one 96 well plate (110 reactions): (check your concentrations before)

  • 4433 µl millipore water

  • 550 µl 10xPCR buffer

  • 330 µl MgCl2 50 mM

  • 110 µl dNTP 10 mM

  • 27.5 µl forward Primer 100 µmol

  • 27.5 µl reverse Primer 100 µmol

  • 22 µl Taq (5 U/µl)

    For the documentation take 5 µl and load it on a 1% agarose gel after the PCR.

Mix the following in vitro transcription mix into a PP-Microplate, 96 well U-shape (1 reaction => 10 µl):

Example for one 96 well plate (110 reactions): (check your concentrations before)

    • 445.5 µl DEPC-water

    • 110 µl Transcription buffer (10xTRB frozen stock)

    • 110 µl ACG nucleotides (each at 4mM in stock solution)

    • 27.5 µl digUTP Mix (4 mM stock)

    • 55 µl DTT (200 mM stock)

    • 22 µl RNA-Polymerase (50 U/µl) (30 Units)


      1. Mix together and aliquot 7µl into each well of the reaction plate.

      2. Then add 3 µl of PCR product to appropriate well.

      3. Place the reaction plate at 37°C (T7-, T3-Polymerase) for 2.5 h in the oven. (For SP6-Polymerase use 40°C). Cover the plate with a Microseal ’A’ Film and a preheated weight.

      4. Set centrifuge to 4°C and heat the oven to 60°C

      5. Following the incubation, add 1µl of RNase-free DNase I (10 U/µl Roche) and incubate for 25 min at 37°C.

      6. For one 96 well plate (110 reactions): (add 5 µl to each reaction of this mix):

  • 44 µl glycogen

  • 440 µl 10 M ammonium acetate

  • 66 µl DEPC-water

      1. Mix well.

      2. Add to each reaction 50 µl of ice cold 100% ethanol (stored at –20°C), mix and centrifuge for 45 min at 4°C and 4000 rpm (Eppendorf Centrifuge 5810R).

      3. Remove all of the supernatant from the reaction tubes using paper towel (cover the plate with paper towel and quickly turn the plate up side down), and add 150 µl ice cold 70% ethanol to each well (stored at –20°C).

      4. Centrifuge tubes for 10 min at 4°C and 4000 rpm.

      5. Remove supernatant as before and place in the fume cupboard to remove remaining ethanol.

      6. Add 50 µl of hydrolyzis buffer, cover the plate with a Microseal ’A’ Film and leave at 60°C for 10 min. Do not mix. After the incubation time store the plate at -20°C.

      7. For the documentation take 5µl and load it on a 1% agarose gel.



Primary Screen Whole Mount ”In Situ Hybridization
with 9.5 dpc to 11.0 dpc Mouse Embryo“

    Day 1: Hybridization

    All tools for this protocol are made by the institutes’ workshop (netwells, netwell reagent tray, netwell tray, probe plate, 104-position plate). We are using the BioLane HTI washing machine from Hölle & Hüttner AG in a version adapted to our needs and equipments.

  • Wash the netwells with millipore water and place them into the netwell tray, fitting on the netwell reagent tray with sterilized tweezers.

  • Fill the reagent tray with approx. 150 ml preheated Hyb solution.

  • Sort the right number of prehybridized embryos into each netwell with a sterile pasteur pipette or with a sterile spoon. For each netwell use 2x 9.5 dpc and 2x 11.0 dpc.

  • Keep the embryo plate at 68°C for 30 min before transferring the netwells to the probe plate.

  • Prepare a handmade 104-position plate with 800 µl Hyb solution per each well and add about 200-300 ng/ml hydrolyzed probe. Mix well and denature the probes at 80°C for 20 min (place the plate in a plastic box and let it swim in a water bath)

  • After the denaturation transfer the pre-heated embryos to the probe plate. Watch out for air bubbles and keep the plate in a humid chamber at 68°C overnight (approx. 16 h). Use a suitable oven, which has a rocking function (Binder-BFED053). Keep the netwell reagent tray with the used Hyb solution in it for next day’s first washing step at 68°C.

  • To prepare next day’s washing steps warm up solution Sol.1, Sol.3T and DEPC-water at 68°C overnight.

    Day 2: Post-hybridization washes:

  • Start the BioLane HTI machine: Flush the machine with pre-heated DEPC-water using a programmed washing programme. (Put all pipes into the DEPC bottle at once)

  1. 1 Delay

  2. 2 Dummy

  3. 8 p3w10 70°C 1 minute position 03 waste 10 volume V5 agitation A5

  4. 9 p4w10 70°C 1 minute position 04 waste 10 volume V5 agitation A5

  5. 10 p5w10 70°C 1 minute position 05 waste 10 volume V5 agitation A5

  6. 11 p6w10 70°C 1 minute position 06 waste 10 volume V5 agitation A5

  7. 12 p8w10 70°C 1 minute position 08 waste 10 volume V5 agitation A5

  8. 13 p7w9 70°C 1 minute position 07 waste 09 volume V5 agitation A5

  9. 0 end of process

  • Transfer the netwell tray from the probe plate back to the Hyb solution plate from day 1, place plate in the oven at 68°C for 5 min. Exchange Hyb and wash again for further 30 min.

  • To pump out the DEPC-water from the machine press F1 and start.

In situ hybridization solution positions at the BioLane HTI machine:

    1. Free

    2. Over pressure, only for special container

    3. TBST

    4. NTT

    5. Antibody solution

    6. 10% lamb serum

    7. Sol.1

    8. Sol.3T

    9. Waste for formamide solutions

    10. Waste for water-based solutions

  • Select the programmed ISH-Programme using the enter key.

  1. 32 Delay 4°C 0 min position 09 waste 09 volume V5 agitation A0

  2. 33 Dummy 21°C 0 min position 09 waste 09 volume V5 agitation A0

  3. 34 Sol1-45 69°C 45 min position 07 waste 09 volume V5 agitation A5 Sol1

  4. 34 Sol1-45 69°C 45 min position 07 waste 09 volume V5 agitation A5 Sol1

  5. 38 Sol3T-45 69°C 45 min position 08 waste 09 volume V5 agitation A5 Sol3T

  6. 38 Sol3T-45 69°C 45 min position 08 waste 09 volume V5 agitation A5 Sol3T

  7. 43 Sol3T-60 69°C 60 min position 08 waste 09 volume V5 agitation A5 Sol3T

  8. 43 Sol3T-60 69°C 60 min position 08 waste 09 volume V5 agitation A5 Sol3T

  9. 47 TBST-15 21°C 15 min position 03 waste 10 volume V5 agitation A5 TBST

  10. 47 TBST-15 21°C 15 min position 03 waste 10 volume V5 agitation A5 TBST

  11. 47 TBST-15 21°C 15 min position 03 waste 10 volume V5 agitation A5 TBST

  12. 52 LS-3h 21°C 180 min position 06 waste 10 volume V5 agitation A5 10% lamb serum

  13. 53 AB-10h 12°C 600 min position 05 waste 10 volume V5 agitation A5 AK

  14. 47 TBST-15 21°C 15 min position 03 waste 10 volume V5 agitation A5 TBST

  15. 47 TBST-15 21°C 15 min position 03 waste 10 volume V5 agitation A5 TBST

  16. 60 TBST-30 21°C 30 min position 03 waste 10 volume V5 agitation A5 TBST

  17. 60 TBST-30 21°C 30 min position 03 waste10 volume V5 agitation A5 TBST

  18. 63 TBST-60 21°C 60 min position 03 waste 10 volume V5 agitation A5 TBST

  19. 65 TBST-2h 18°C 120 min position 03 waste 10 volume V5 agitation A5 TBST

  20. 65 TBST-2h 18°C 120 min position 03 waste 10 volume V5 agitation A5 TBST

  21. 67 TBST-3h 15°C 180 min position 03 waste 10 volume V5 agitation A5 TBST

  22. 67 TBST-3h 15°C 180 min position 03 waste 10 volume V5 agitation A5 TBST

  23. 67 TBST-3h 15°C 180 min position 03 waste 10 volume V5 agitation A5 TBST

  24. 71 TBST-4h 12°C 240 min position 03 waste 10 volume V5 agitation A5 TBST

  25. 71 TBST-4h 12°C 240 min position 03 waste 10 volume V5 agitation A5 TBST

  26. 71 TBST-4h 12°C 240 min position 03 waste 10 volume V5 agitation A5 TBST

  27. 63 TBST-60 21°C 60 min position 03 waste 10 volume V5 agitation A5 TBST

  28. 75 NTT-10 21°C 15 min position 04 waste 10 volume V5 agitation A5 NTT

  29. 75 NTT-10 21°C 15 min position 04 waste 10 volume V5 agitation A5 NTT

  30. 75 NTT-10 21°C 15 min position 04 waste 10 volume V5 agitation A5 NTT

  31. 0 end of process

  • When Sol.1 is pumped into the machine tray, place the embryo netwell tray onto it and close the lid with screws and cover it with polystyrene.

    Needed volume of solutions for one plate:

  • DEPC-water: 1000 ml

  • Sol.1: 360 ml

  • Sol.3T: 720 ml

  • TBST: 3000 ml

  • NTT: 840 ml

  • 10% lamb serum: 180 ml

  • Antibody solution: 180 ml

  • Hyb: 360 ml

  • NTMT: 1000 ml

  • For the staining solution: 1350 µl NBT; 1050 µl BCIP

  • PBST: 400 ml

  • 4%PFA/PBST: 180 ml

    Preparation of 10% lamb serum blocking reagent: 18 ml heat-inactivated lamb serum (stored aliquots are

    at -20°C) + 162 ml TBST-DEPC (using a 500 ml bottle).

    Preparation of the antibody solution: Mix 4x 12 ml TBST-DEPC with embryo powder (a tip of a small spoon) directly into a 15 ml Falcon tube for inactivation at 70°C (water bath) for 30 min. Afterwards cool it down on ice and add 120 µl lamb serum as well as 22.5 µl α-Dig-AP (Roche) to each tube. Wrap it with tinfoil and roll it at 4°C for at least 1 h. Spin down the embryo powder at 4000 rpm and 4°C for 10 min (Eppendorf Centrifuge 5810R) and add the antibody solution to the 1% lamb serum/TBST BioLane HTI chilled container (1.32ml lamb serum + 130.68ml TBST). Mix well.

    Day 3: Post-antibody-washes

    See programmed ISH-Programme

    Day 4: Staining

  • After finishing the ISH-Programme, wash the embryos twice with NTMT in a handmade netwell reagent tray for 20 min (approx. 180 ml).

  • During the NTMT washing steps prepare the staining solution:

300 ml NTMT

1350 µl ml NBT -> 75 µg/µl nitro blue tetrazolium-saltin 70% (v/v) dimethylformamide (toxic)

1050 µl BCIP -> 50 µg/µl 5-bromo-4-chloro-3-indolyl-phosphate toluidinium-salt in 100% dimethylformamide

Keep it dark and sterilize the staining solution with a 0.22 µm vacuum filter.

Use 150 ml staining solution for one netwell reagent tray.

  • Transfer the embryos to the staining tray and rock it at RT in the dark for 15 min. Afterwards, keep it in the dark and check the staining reaction periodically under a binocular. If the staining reaction is not completed at the end of the day, apply fresh staining solution and keep the plate at 4°C overnight.

  • To stop the staining reaction wash once with NTT and several times with PBST. Single embryos can be transferred from NTT back to the staining solution if staining needs to be prolonged.

  • It is recommendable to fix the stained embryos with 4% PFA/PBST overnight to avoid further staining

    (96x 800 µl 4% PFA/PBST -> 2x 48 well plates)

  • If needed embryos can be clarified with PBST/50% formamide.

  • Store embryos in the dark in 4% PFA/PBST at 4°C. Embryos in PBST/50% formamide can be stored at

    –20°C.

Cleaning the Biolane machine:

Millipore water und 70% ethanol/millipore water

  1. 1 Delay

  2. 2 Dummy

  3. 24 p1w10

  4. 25 p2w10

  5. 26 p3w10 21°C 1min position 03 waste 10 volume V5 agitation A5

  6. 27 p4w10 21°C 1 min position 04 waste 10 volume V5 agitation A5

  7. 28 p5w10 21°C 1 min position 05 waste 10 volume V5 agitation A5

  8. 29 p6w10 21°C 1 min position 06 waste 10 volume V5 agitation A5

  9. 30 p7w10 21°C 1 min position 07 waste 10 volume V5 agitation A5

  10. 31 p8w10 21°C 1 min position 08 waste 10 volume V5 agitation A5

  11. 0 end of process



Preparation of Probes for “Time Course-In Situ Hybridization” Involving In vitro Transcription


This Process must be RNase free! Use clean gloves, sterile solutions and RNA solutions and chemicals.

For longer-term storage of probes after hydrolyzis, re precipitate following steps 7-12 and resuspend pellet in DEPC-water.

In the primary screen candidates have been selected. Prepare a MIDI-plasmid preparation of each candidate. We are using the Jetstar 2.0 plasmid purification Kid.


Mix the following PCR mix (the following is for 1 reaction => 50 µl):

Example: (check your concentrations before)

        • 36.5 µl DEPC-water

        • 5 µl 10xPCR buffer

        • 1.5 µl MgCl2 50 mM

        • 1 µl dNTP 10 mM

        • 0.25 µl forward-Primer 100 µmol

        • 0.25 µl reverse-Primer 100 µmol

        • 0.5 µl Taq (5 U/µl)

        • 5 µl DNA-tamplate 1 ng/µl

For the documentation take 5 µl and load it on a 1% agarose gel after the PCR run


Mix the following in vitro transcription mix (1 reaction => 21 µl):

Example: (check your concentrations before)

    • 12.15 µl DEPC-water

    • 3 µl Transcription buffer (10xTRB frozen stock)

    • 3 µl ACG nucleotides (each at 4 mM in stock solution)

    • 0.75 µl digUTP Mix (4 mM stock)

    • 1.5 µl DTT (200 mM stock)

    • 0.6 µl RNA-Polymerase (50 U/µl) (30 Units)


      1. Mix together and add 21 µl into each reaction tube (1.5ml Eppendorf tube).

      2. Then add 9 µl of PCR product to appropriate well.

      3. Place the reaction tubes at 37°C (T7-, T3-Polymerase) for 2.5 h. (For SP6-Polymerase use 40°C)

      4. Set centrifuge to 4°C and heat the water bath to 60°C

      5. Following the incubation, add 3 µl of RNase-free DNase I (10 U/µl Roche) and incubate for 15 min at 37°C.

      6. Add to each reaction:

  • 1.2 µl glycogen

  • 12 µl 10 M ammonium acetate

      1. Mix well.

      2. Add to each reaction 150 µl of ice cold 100% ethanol (stored at –20°C), mix and centrifuge for 30 min at 4°C and 13200 rpm (Eppendorf Centrifuge 5415R).

      3. Remove all of the supernatant from the reaction tubes, and add to each pellet 150 µl ice cold 70% ethanol (stored at –20°C).

      4. Centrifuge tubes for 10 min at 4°C and 13200 rpm.

      5. Remove supernatant and place in the fume cupboard to remove remaining ethanol.

      6. Re-suspend the pellet in 30 µl of hydrolyzis buffer and leave at 60°C for the calculated time:

PCR kb - desired size kb / 0.11*PCR kb* desired size kb (Our desired fragment size is 1kb)

      1. Place on ice to cool and repeat the protocol from step 6. (Without Glycogen)

      2. Dissolve the final pellet in 100 µl DEPC-water and store at -20°C.

      3. For the documentation take 5 µl and load it on a 1% agarose gel.

    Time Course “Whole Mount” In Situ Hybridization
    Of 8.5-11.5 dpc Mouse Embryos

    Day 1: Hybridization

  • For this protocol use 12-well plates from Costar. Fill each well with 2 ml Hyb-Solution (preheated to 68°C), place a clean netwell into each well (Cornstar, 15 mm diameter, 74 µm mesh). Sort the right number of prehybridized embryos into each netwell with a sterile pasteur pipette or with a sterile spoon. For each probe/well use 2x 8.5 dpc, 5x 9.5 dpc, 2x 10.5 dpc, 2x 11.5 dpc embryos.

  • Keep the embryos in netwells at 68°C for 30 min before transferring the netwells to the probe plate.

  • Prepare a fresh pre-heated 12-well plate with 2 ml Hyb solution and add about 200-300 ng/ml hydrolyzed probe. Mix well and denature the probes at 80°C for 10 min (place the 12-well plate in a plastic box and let it swim in a water bath)

  • Right after the denaturation, transfer the netwells with embryos to the probe plate. Watch out for air bubbles and keep the plate in a humid chamber at 68°C overnight (approx. 16 h). Use a suitable oven, which has a rocking function (Binder BFED 053). Keep the 12-well plate with the used Hyb solution in it for next day’s first washing step at 68°C.

  • To prepare next day’s washing steps warm up solution Sol.1 and Sol.3T at 68°C overnight.

    Day 2: Post-hybridization washes:

  • Transfer the netwells from the probe plate back to the 12-well Hyb solution plate from day 1, place plate in the oven at 68°C for 30 min.

  • From now on, use washing trays for the following washing steps (Corning, netwell reagent tray and netwell tray).

  • Wash twice with Sol.1 at 68°C for 30 min (approx. 90 ml)

  • Wash twice with Sol.3T at 68°C for 30 min and twice for 60 min (approx. 90 ml).

    During the last Sol.3T washing step start preparing the antibody solution.

  • Wash three times with TBST-DEPC rocking at RT for 15 min. While the second washing step is running flush the netwells gently with TBST with a sterile pasteur pipette to remove rests of formamide.

  • Prepare the blocking reagent, which prepares the embryo for the α-Dig-AP (Roche). Use a fresh 12-well plate and fill 2ml 10% lamb serum/TBST into each well (2.5 ml deactivated lamb serum + 22.5 ml TBST). Transfer the netwells into the blocking solution and incubate at RT for 2-3 h.

  • Antibody preparation (for one 12-well plate): Prepare 19 ml 1% lamb serum/TBST (19 ml TBST + 190 µl lamb serum) using a 50 ml Falcon tube. Mix 6ml TBST-DEPC with embryo powder (a tip of a small spoon) directly into a 15ml Falcon tube for inactivation at 70°C (water bath) for 30 min. Afterwards, cool it down on ice and add 60 µl lamb serum as well as 12.5 µl α-Dig-AP (Roche). Wrap it with tinfoil and roll it at 4°C for at least 1 h. Spin down the embryo powder at 4000 rpm and 4°C for 10 min (Eppendorf Centrifuge 5810R) and add the antibody solution to the 1% lamb serum/TBST tube. Mix well. Use a fresh 12-well plate and fill 2ml antibody solution into each well and keep it in the dark at 4°C before and during incubation. After the blocking step transfer netwells from the blocking solution into the antibody solution. Watch out for air bubbles and incubate the plate rocking at 4°C overnight (approx. 10 h).

    Day 3: Post-antibody-washes

  • Wash embryos twice for 15 min, 30 min and at least six times for 1 h with approx. 90 ml TBST prepared with millipore water at RT reusing the netwell reagent tray. Another washing step with TBST at 4°C overnight can reduce background staining in the older embryos and foetuses.

    Day 4: Staining

  • Wash embryos four times for 15 min with approx. 90 ml NTMT at RT (flush the netwells gently with NTMT to remove all TBST as this would spoil the staining reaction)

  • For one 12-well plate prepare 25 ml NTMT with 112.5 µl NBT and 87.5 µl BCIP (2 ml per well) in a 50 ml Falcon tube on ice sterilize the staining solution with a 0,45 µm syringe filter and keep it dark.

  • Transfer embryo netwells to the staining reaction plate and rock it in the dark at RT for 15 min. Afterwards, keep it in the dark and check the staining reaction periodically under a binocular. If the staining reaction is not completed at the end of the day, apply fresh staining solution and keep the plate at 4°C overnight.

  • To stop the staining reaction wash once with NTT and several of times with PBST. Single embryos can be transferred from NTT back to the staining solution if staining needs to be prolonged.

  • It is recommendable to fix the stained embryos with 4% PFA/PBST overnight to avoid further staining.

  • If needed, you can clarify embryos with PBST/50% formamide.

  • Store embryos in the dark in 4% PFA/PBST at 4°C. Embryos in PBST/50% formamide can be stored at

    –20°C.

Solutions:


To treat solutions with DEPC, add 1/5.000 vol. DEPC, incubate at least 2 h at 37°C or overnight at RT and autoclave. DEPC is toxic. Wear gloves! Do not inhale vapours! Keep stocks separate for RNsae-sensitive steps


PBS 8 g NaCl
0.2 g KCl
1.44 g Na2HPO4
0.24 g KH2PO4
add water to 1l
pH should be 7.4, otherwise adjust with HCl/NaOH
can be prepared as 10-times stock solution, in this case pH should be 6.8

DEPC-PBST DEPC-PBS + 0.1% Tween-20
add Tween-20 after DEPC treatment and autoclaving!

Prot.K (common stock -20°C)
10 µg/µl Proteinase K (Roche) in DEPC-water
discard aliquots after use! (Rests of aliquots can be used for cleaning the netwells)

Glycin (common stock -20°C)
200 µg/µl glycine in DEPC-water

Glut. (common stock -20°C)
25% glutaraldehyde (Sigma)
discard aliquots after use!

4% PFA/PBST dissolve 4 g paraformaldehyde powder in 100 ml PBST at 60°C (approx. 1h) cool on ice; filter. Aliquot and store at –20°C.

Hyb 250 ml formamide
125 ml 20x DEPC-SSC pH 5.0
25 ml 20% SDS (stock solution)
500 µl yeast RNA (stock solution)
500 µl heparin (stock solution)
add DEPC-water to 500 ml
store at -20°C

20x DEPC-SSC pH 5.0 88.23 g Na3-citrate-dihydrate
175.32 g NaCl
add DEPC-water to 1l
adjust pH 5.0 with citric acid
DEPC treatment

20% SDS 200 g SDS
add DEPC-water to 1l and dissolve by heating
filter sterilize, do not autoclave!

yeast RNA (common stock)
50 µg/µl yeast RNA in DEPC-water

Heparin (common stock)
50 µg/µl heparin in DEPC-water

Sol.1 500 ml formamide
250 ml 20x SSC pH 5.0
50 ml 20% SDS
add DEPC-water to 1l
store at -20°C

Sol.3T 500 ml formamide
100 ml 20x SSC pH 5.0
1 ml Tween-20
add DEPC-water to 1l
store at -20°C

10xTBST 81.8 g NaCl
2 g KCl
30.3 g Tris-base
add water to 900 ml and dissolve
use 37% HCl to adjust pH 7.5 (approx. 30ml)
add water to 1l
autoclave and add 10 ml Tween-20

Lamb serum (common stock)
Lamb serum heat inactivated for 30 min at 56°C

NTMT 429.5 ml water
50 ml 1 M Tris-HCl pH 9.5
10 ml 5 M NaCl
10 ml 2.5 M MgCl2
500 µl Tween-20
always freshly prepared

NTT 439.5 ml water
50 ml 1 M Tris-HCl pH 9.5
10 ml 5 M NaCl
500 µl Tween-20

BCIP 50 µg/µl 5-bromo-4-chloro-3-indolyl-phosphat toluidinium-salt in 100% dimethylformamide
dimethylformamide is toxic!

NBT 75 µg/µl nitro blue tetrazolium salt in 70% (v/v) dimethylformamide
dimethylformamid is toxic!

  • Netwell cleaning: Flush netwells with millipore water after usage. Keep them in 3% H2O2 / millipore water. If necessary, wash with Prot K (1 µg/ml) / 1% SDS overnight and keep in 3% H2O2 / millipore water until the next usage. Before using the netwells, wash at least twice with millipore water.