Protocols/Methods & Materials

Biolistic Gene Gun

DRG Cultures

Genotyping

In-Situ Hybridisation

Whole Cell Perforated Patch Recording

TFFC: Mr T's Fruit Cake recipe

Biolistic Gene Gun

Materials: Microcarriers 1.6 m gold particles, Bio-Rad cat no: 165-2264; Macrocarriers, Bio-Rad cat no: 165-2335; Rupture disks, Bio-Rad cat no: 165-2326; Stopping screens, Bio-Rad cat no: 165-2336; Ethanol ( HPLC grade filtered with 0.22 m); Isopropanol ( HPLC grade filtered with 0.22 m); Water ( filtered with 0.22 m); Calcium chloride ( filtered with 0.22 m); 50 % glycerol ( filter sterilised 0.22 M); DRG cells are all concentrated in the centre of the dish. Resuspend pellet in approx 50-100l media and let the cells settle for 4 hrs to overnight before flooding the dish.

Preparing gold particles: Insert plastic macrocarriers into metal holders, wrap individually and autoclave. Autoclave 3 stop screens per individual wrap. Preparing gold microcarriers for 6 bombardments. Weigh out 3 mg into a sterile eppendorf. Add 1 ml of 70% ethanol. Vortex for 4-5 mins vigorously to wash all the particles. Pellet by spinning for 30 s at 13000 rpm. Remove the supernantant. Add 1 ml of sterile water and vortex for 1 min. Pellet as above and discard the supernantant. Repeat 2 times. Remove all the liquid and add 50 l of sterile 50% glycerol. This will give you a concentration of 60 mg/ml gold particles. Store in a tube containing dessicant until required. Use on the day of transfection.

Coat gold particles with DNA: For 6 bombardments, take gold particles and add in order, 5 l of each type of DNA ( 1 ug/l), 50 l of 2.5 M CaCl2, and 50 l of 0.1 M spermadine ( base free, TC grade). Vortex continuously for 15 mins. Leave for 3-4 mins so the gold particles settle at the bottom of the tube. Remove the supernatant. Add 140 l of 70 % Ethanol and vortex for 1 min. Leave to settle and remove the supernantant. Add 48 l of 100% ethanol and resuspend the gold particles by vortexing for 3 min. Place the macrocarriers into pertri dishes containing silica. Transfer 8 l of suspended gold onto the centre of the plastic in each macrocarrier, smear around the plastic with the tip of a pipette and dry inside a TC hood. Use within 2 hours.

Shooting the cells: Shoot a blank with stop disks and rupture disks only before shooting the cells. Clean the inside of the gene gun chamber with 70% ethanol and allow to dry in a closed atmosphere. Dip the rupture disk into 70% isopropanol, remove excess by flicking and place in the centre of the retaining cap. ( Rupture disk holder) Screw the cap anticlockwise into the chamber and tighten with the appropriate tool. Assemble microcarrier launch unit. Plug in the Biolistic unit and vacuum pump. Open the helium valve to achieve at least 500 psi output pressure. Switch on the vacuum pump at the plug as well as the biolistic unit. Turn the chamber on. Gently remove the media from the cells with a syringe ( retain the media within the syringe to re-feed the cells) and place them on the clear shelf, cells in the middle on the shelf below the launch unit. Shut the door. Swith to VAC position and wait until the vacuum level in the chamber is 6-7 Hg inches then immediately switch to HOLD position. Press the fire button and wait for positive pressure to build up to 450 psi. Immediately switch VAC to VENT and as soon as the vacuum has been released remove the cells and replace the media. Repeat for each bombardment. Change the media on the cells.

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DRG Cultures

Protocol will be available shortly.

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Genotyping (Mouse)

Isolation of genomic DNA from mouse tails:

1) Add 750 l of buffer (50 mM Tris pH 8.0, 100 mM EDTA, 100 mM NaCl, 1% SDS) and 20 l of proteinase K solution (14 mg/ml, Boehringer Mannheim)to 0.7 -1 cm of mouse tail and mix.

2) Incubate at 55C overnight.

3) After incubation add 250 l saturated NaCl. Mix by hand or vortex.

4) Centrifuge at top speed for 10 mins.

5) Transfer 750 l supernatant into fresh tube, to it add 750 l isopropanol and mix. Pellet DNA by spining for 5-10 mins in a microfuge.

6) Discard supernatant. Add 1 ml 70% ethanol. Shake well.

7) Dry DNA in a vacuum centrifuge for 3-5 mins or air dry for 15 mins.

8) Resuspend DNA in 100-200 l of water.

PCR of mouse genomic DNA:

1) Add 2.5 l of 10X Mg-free Taq DNA polymerase buffer (Promega), 0.5 l Taq DNA polymerase, 1.5 l of 25 mN MgCl2, 1 l of 5 mM dNTP, 1 l DNA, and appropriate amount of primers, bring final volume to 25 l with water.

2) PCR using the following cycles - 1 cycle of 94C for 4 mins, 35 cycles of 94C 1 min, 58C 1 min, 72C 1.5 min. Note: Optimal number of cycles and temperatures differ for different primers and PCR machines.

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In-Situ Hybridisation

IN-SITU: DIG PROBES DETECTING mRNA

Section Preparation:

1) Coat slides with 2% APES solution.

2) Cut 10 m sections from DRG and put them on the coated slides.

3) Air-dry the sections for 20 min to 3 hours max. After 3 hours RNA begins to degrade. Slides can be stored at -20C in a sealed box containing desiccants.

Fixation:

1) Cool slide chamber on ice. Fix the sections in cold 4% paraformaldehyde for 10 mins max. Increased fixation decreases sensitivity. Fixation at 0-4C will inhibit endogenous ribonucleases.

2) Wash 3 times in cold PBS for up to 2 min shaking gently.

3) Place slide rack into a stirring solution of Triethanolamine/acetic anhydride for 10 min, RT. Use a rod to suspend the rack above the stirrer in a small chamber.

4) Wash the slides 3 times in PBS at RT as above. The slides can be kept up to 3 hours in PBS before proceeding to the next stage. Do not allow the slides to dry out.

Pre-hybridisation:

1) Place a 50% formamide/4xSSC mix in the bottom of a humid box (25 ml low grade formamide, 10 ml 20xSSC, 15 ml water).

2) Take the slides from the PBS and wipe dry the bottom side and opaque portion of the glass. Place the slide section side up in the box. 3) Add 700-800 l hybridisation buffer to each slide, cover with the lid and leave for 1-6 hours at RT.

Probe:

1) Take 10-300 ng (/ml) of probe RNA and add to 100 l of hybridisation buffer.

2) Place at 80-90C for 5 minutes or 1 min at 100C to denature. Place on ice immediately.

3) Add 900 l cold hybridisation buffer to the mix and leave on ice until required.

Hybridisation:

1) Remove the buffer from slide by flicking. Dry the underside of the slide.

2) Add 160 l of RNA and hybridisation buffer to each slide. Ensure all the sections are covered.

3) Cut slide-shaped pieces from plastic hybridisation bags and gently lay them over the slides. Avoid bubbles. Put lid on, seal the box and place at 55-66C for 16-17 hours. Ensure the chamber still contains the formamide mix.

Washes:

1) Place the slides in 2xSSC so that the plastic cover lifts off.

2) Rack the slides and wash 4 times in 2xSSC, 65-72C, gently shaking for 10 min.

3) Wash in 65-72C 2xSSC, shaking for 45 min at 65-72C.

4) Wash in 0.1xSSC, shaking for 1 hour at 65-72C. Cool for 30 minutes at RT. 5. Wash in RT 0.1-0.2xSSC, shaking for 10 min.

Detection:

1) Place rack in B1 solution for 10 min, shaking.

2) Place in fresh B2 solution for 1 hour, lie flat in the box.

3) Add anti-DIG to solution B2 (3-1:1500) and add 160-250 l per slide, cover with plastic and leave for 1.5 hours flat at RT. Place D.H2O in the bottom of the humid box and cover with lid.

4) Place in solution B3 for 5 min. Place slides back in the box, flat after cleaning the underside of the slide.

5) Add 150-200 l B4 solution per slide, cover with plastic and leave for 1 hour to overnight in the dark.

6) Check the slides under the microscope periodically and once the reaction is complete, stop the reaction with PBS or B5. Keep the slides in PBS at 4C .

Solutions:

1) TEA/AA: Triethanolamine 3.5 ml; distilled water 300 ml; Acetic anhydride 750 l (add while solution is stirring).

2) Hybridisation Buffer: Final volume 100 ml; 100% mol grade formamide 50 ml; 20xSSC 20 ml; 50x Denharts soln 4 ml; 10 mg/ml tRNA (clean) 0.5ml; 5mg/ml denatured salmon sperm DNA 3 ml; Sterile water 22.5 ml. Formamide should be pH 8-10. Freeze on arrival at -20C. If not the pH will drop as it deionises.

3) B1: Final volume 1 l; 1 M Tris pH 7.5 100 ml; NaCl 8.76 g. bring up to 1 l with distilled water.

4) B2: 2% sheep serum in B1 solution.

5) B3: Final volume 500 ml; 1 M Tris 50 ml; NaCl 2.9 g; 5 M MgCl2 5 ml; distilled water to bring up to 500 ml. Adjust pH 9.5 by adding 340 l conc HCl.

6) B4: B3 1 ml; NBT 4.5 l; BCIP 3.5 l; Levamisole (5 mg/ml) 25 l; protect from light.

In Vitro Transcription for In Situ Hybridisation

1) Linearise plasmid DNA (3-5 g) with a suitable restriction enzyme at 37C for 2 hr.

2) Add 1 ul tRNA (10 g) and STE, then extract with phenol/chloroform.

3) Precipitate with 1/10 vol sodium acetate and 2.5 vols ethanol.

4) Dissolve pellet with 5 l H2O.

5) Add following solutions in sequence: 10 l H2O, 2 l 10x transcription buffer (BM), 2 ul 10x NTP labelling mix (BM), 5 l DNA temple, 0.3 l RNase inhibitor - RNasin (BM). Mix. Add 1ul T7 or Sp6 polymerase (BM) and put at 37C immediately. Leave 1 hr.

6) Add 0.5 l DNaseI, keep at 37C 30-45 min.

7) Add 130 l STE , 75 l acid-phenol, and 75 l chloroform. Mix and spin. DNA enters the organic phase provided it is acidic enough, whereas the RNA stays in the aqueous phase.

8) Collect aqueous layer, add 1/10 vol 3 M Sodium Acetate pH 4.0.

9) Add 150 l isopropanol. Mix and keep at -20C at least 1.5 hr.

10) Spin 20 min at 4C. Wash with 70% ethanol.

11) Dry sample, dissolve with 20 l H2O.

12) Take 0.8 l sample and add 5 l stop solution. Heat at 70C, 5 min, and then put on ice.

13) Check the transcript quality on 1% agarose gel.

In Situ Hybridisation:

Hybridisation mix: 50% formamide, 4x SSC, 2x Denhardt's solution, 50 g/ml tRNA, 150 g/ml ssDNA (denatured).

Sample preparation:

1) Cut sections (10 m thick) then stick them on Vectabond coated slides.

2) Air dry at room temp. for about 20 min but never longer than 3 hr to prevent degradation of mRNA. (Dried section can be stored at -20C.)

3) Fix sections in cold 4% paraformaldehyde (in PBS) 10 min on ice.

4) Wash three times with PBS, then acetylated by triethanolamine (3.5 ml in 300 ml H2O) and 750 l acetic anhydride for 10 mins.

5) Wash sections three times by PBS.

Hybridisation:

1) Add 400-800 l hybridisation mix to each slide, then put them in humidified chamber at room temp at least 1 hr.

2) Add 0.3 l dig-labelled probe in 1 ml hybridisation mix (300 ng/ml). Denature at 80C 5 min.
3) Remove hybridisation mix from slides, then add 160 l probe/hybridisation mix. Incubate at 66C overnight.

4) Wash slides with 2xSSC shaking 10 min at 72C, four times.

5) Put it at 72C (pre-warmed) 2xSSC for 30 mins, then 72C 0.1xSSC for 1 hr.

6) Wash with 0.2xSSC 10 mins at room temp. The following steps are handled at room temp.

7) Put in B1 solution for 10 mins (0.1 M Tris pH7.5, 0.15 M NaCl).

8) Put in B2 solution for 1 hr.

9) Add anti-Dig in B2 (1500x dilution) one and half hours.

10) Put in B3 solution for 5 mins (0.1 M Tris, 0.1 M NaCl, 50 mM MgCl2 pH 9.5).

11) Add 150 l B4 solution to each slide, then put in dark. (B4: 4.5 l NBT, 3.5 l BCIP, 25 l 5mg/ml levamisole in 1 ml B3).

12) Add B5 solution to stop staining (10 mM Tris pH 8.0, 1 mM EDTA).

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Whole Cell Perforated Patch Recording

For this protocol, please refer to "
The Axon guide - Chapter 5: Advanced Methods in Electrophysiology Recording from Perforated Patches and Perforated Vesicles."

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Mr T's Fruit Cake Recipe

The original recipe didn't include lemon juice, but I put the juice in because I wasn't sure what "zest" was. I saved the rum that the fruit had been in and poured it over the cake after cooking.....

150g mixed fruit

100g glace cherries

150g dried apples, pears or apricots in ~ 1cm cubes 100ml rum 250g marzipan 50g ground almonds zest and juice of 1 lemon 175g plain flour 100g caster sugar 100g butter

2 large eggs

20cm cake tin, buttered and lined so that the parchment comes 10cm above the rim

Mix mixed fruit, glace cherries and dried apples/pears/apricots in a large bowl, cover with the rum. Dice the marzipan and put it in the freezer. Leave both to soak and freeze.

Preheat oven to 140oC. Beat together almonds, lemon zest and juice, sugar, flour, butter and eggs. Add drained fruit and frozen marzipan. Put the mixture into the tin, levelling the surface, with a slight indent in the middle.

Bake for 2 - 2 1/2 hours, or until skewer comes out clean.

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