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Poulin Lab

Protocols

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[-] Freezing Buffer (2x)

For 100ml

  • NaCl(1M) - 10ml
  • KH2PO4(1M) (pH6) - 5ml
  • Glycerol - 30ml
  • MgSO4(0.1M) - 300µl
  1. Mix well and make up to final volume 100ml with Milli-Q water.
  2. Filter sterilise.
  3. Aliquot into 15ml tubes date, and label.

[-] KH2PO4 Buffer (1M, pH6)

For 1 litre

  • KH2PO4 - 136g
  1. Dissolve the above chemicals in 800ml Milli-Q water.
  2. pH to 6 with KOH pellets.
  3. Make up to 1 litre, aliquot and autoclave.

[-] Bleaching in a Drop

  1. Place a small drop of bleaching solution in the middle of a NGM plate containing food.
  2. Pick ~5 -10 mothers with eggs inside and place them in the bleach drop.
  3. Let the bleach drop dry.
  4. Add another drop if the mothers have not disintegrated. (NOTE: do not add more than 3 drops of bleach as you may kill the eggs).
  5. Let the bleach drop dry.
  6. The next day the L1 larvae should have hatched.

[-] Bleaching Solution

  • NaOH(10M) - 1ml
  • Sodium Hypochlorite (bleach) - 3ml
  • Distilled Water - 10ml
  1. Mix well.

[-] Bleaching Worms

  1. Rinse adult gravid worms from the NGM plate(s) using M9 buffer. Pipette worms in to a 15ml tube (NOTE: rinse the pipette tip with M9 buffer + Tween-20 before sucking up the worms. This prevents the worms from sticking to the side of the tip.
  2. Spin down the worms by centrifuging at 1150xg for 1.5 mins.
  3. Aspirate the supernatant. Be careful not to knock the tube as you will aspirate your worms!!
  4. To each tube of worms add 1-2ml bleaching solution (see bleaching solution recipe).
  5. Vortex tube until all the mothers have dissolved. This should be between 5-7 mins. Do not leave the worms in bleaching solution for more than 7 mins as the eggs will bw affected and will not hatch. After the mothers have dissolved fill up the tube to 15ml with M9. Mix well.
  6. Spin down the worms by centrifuging at 1150xg for 1.5 mins. Aspirate supernatant, and fill up tube again with M9. Be careful not to knock the tube as you will aspirate your worms!!
  7. Repeat the above step 3 more times.

Following the last wash step eggs can be left to hatch either by:

  • Spotting the eggs onto NGM plates without food, remembering to rinse tip with M9 + tween before pipetting. Leave to hatch O/N at 20°C.
  • Next day, wash off the hatched L1’s with M9. Spin down as described above and aspirate the supernatant.
  • Pipette L1’s onto NGM plates with food and place at 20°C.

Or by

  • Filling the 15ml tube with M9 to ~6ml, and then leaving on the rocker O/N.
  • Next day, spin down as described above and aspirate the supernatant.
  • Pipette L1’s onto NGM plates with food and place at 20°C.

[-] Colony PCR

  • NH4 Buffer(10x)
  • MgCl2(50mM)
  • dNTP’s(2mM)
  • Both Forward and Reverse primer(10µM)
  • TAQ (usually Bio-Taq)
  1. Make your PCR mix containing: 1x NH4 buffer, 1.5mm MgCl2, 0.2mm dNTP’s, 0.5µm F and R primer and 0.1U/µl Taq polymerase.
Per reaction example:
  • Water - 19.6µl
  • NH4 buffer(10x) - 3µl
  • MgCl2(50Mm) - 0.9µl
  • dNTP’s(2Mm) - 3µl
  • Forward primer(10µM) - 1.5µl
  • Reverse primer(10µM) - 1 .5µl
  • Taq - 0.5µl
  1. Pipette 30µl of the PCR mix in each 200µl PCR tube.
  2. Pick a single colony from your plate, firstly restreak it onto your master plate using a tip and then place the tip into the tube containing the PCR mix.
  3. Leave the tip in the tube for a few minutes, mix a little and then remove.
  4. Run the PCR for 25 cycles.

[-] Flat Plates

For 1 litre

  • NaCl - 10g
  • Yeast extract - 5g
  • Tryptone - 10g
  • Agar - 15g
  1. Dissolve and mix the above chemicals in 1 litre of Milli-Q water. (NOTE: agar will not dissolve into solution until it has been autoclaved).
  2. Autoclave.
  3. Cool to ~55°C.
  4. Add 1ml of Tetracycline(3x) and 1ml of Carbenicillin(1x).
  5. Mix well.
  6. Pour into flat plates (~50ml). Once set keep wrapped in tin foil as they are light sensitive.

[-] Freezing Worms

Note

  • Each time you freeze a worm strain remember to make an extra ‘test tube’ in order to make sure your worms survive the freezing process and that they are also not contaminated.
  • On average 1 (6cm) plate of starved L1 worms will be enough for one frozen tube of worms.
  1. Wash starved L1 off plates using M9 buffer (remember to rinse tip with M9 + tween), and place in a 15ml tube. Fill tube to 15ml with M9.
  2. Centrifuge the worms for 1.5 mins at 1500xg.
  3. Aspirate the supernatant leaving 250µl buffer per freezing tube. This is done best by marking on the side of the 15ml tube beforehand where 250µl would reach.
  4. Per freezing tube add 250µl of freezing buffer (see freezing buffer recipe).
  5. Mix by pipetting up and down with a tip that has been rinsed with M9 buffer +Tween-20.
  6. Aliquot the 500µl into one 1.5ml screw cap tube.
  7. Freeze the tubes slowly by placing them in polystyrene box in the -80°C freezer.
  8. Thaw the test tube on the bench for a few minutes or until it has completely melted. Pipette the entire 500µl aliquot on to an NGM plate containing food. Leave the plate to dry next to a bunsen with the lid off. A recovery of 35-45% is usually expected.

[-] Growing OP50

Day One

  • Streak out OP50 from either glycerol stock or an old spotted NGM plate onto an LB agar plate. Leave overnight at 37°C.

Day Two

  • Using a tip take a few colonies of the OP50 and inoculate a 250ml duran bottle of LB miller. Leave O/N in the static 37°C incubator.

Day Three

  • The OP50 is now ready for spotting. Aliquot the OP50 into ~10, 15ml tubes and place in the fridge. Remake fresh OP50 every 2 weeks.

[-] LB Miller

For 1 litre

  • NaCl - 10g
  • Yeast extract - 5g
  • Tryptone - 10g
  1. Dissolve the above chemicals in 800ml Milli-Q water.
  2. Once dissolved make up to 1 litre, aliquot and autoclave.

[-] M9 Buffer

  • Na2HPO4.12H2O - 30.27g
  • KH2PO4 - 6g
  • NaCl - 10g
  1. Dissolve the above chemicals in ~1.7 litres of distilled water.
  2. Mix well and make up to final volume of 2 litres.
  3. Aliquot into either 500ml, 250ml and 100ml bottles date, label and autoclave.
    Important: wait until buffer has cooled down.
  4. Add 1M sterile MgSO4 at 100µl/100ml of M9 buffer.
    • add 500µl per 500ml bottle of M9 buffer.
    • add 250µl per 250ml bottle of M9 buffer.
    • add 100µl per 100ml bottle of M9 buffer.

Before using, test the M9 to make sure it is not contaminated by spotting 4 drops onto a NGM plate without food. Once dry place the plate in the 37°C incubator O/N. Next day check for contamination.

[-] Mounting Worms on Microscope Slides

  1. Melt a 2ml aliquot of 2% agarose over a bunsen flame until it becomes viscous (WARNING!! Heat up the aliquot slowly, as heated agar can spit. Hold the tube away from you and wear goggles!!!).
  2. Keep the melted agar in a heat block at 80°C so that it does not solidify.
  3. Add ~100µl of melted 2% agarose to the centre of a microscope slide. Using another microscope slide lightly press down on the agar, so that it forms a flat circular disc. Once the agar has set (~30 secs) remove the top microscope slide and any uneven agar.
  4. Place 2 drops (total volume ~7µl) of 20mM tetramisole (to paralyse the worms) on the agar disc.
  5. Pick the worms you want to mount and place them in the drops of tetramisole.
  6. Place a coverslip carefully over the top so not to get any bubbles.

[-] NGM Plates

For 1 litre

  • NaCl - 3g
  • Peptone - 2.5g
  • Agar - 17g
  1. Dissolve the above chemicals in 975ml Milli-Q water. (Note: agar will not dissolve into solution until it has been autoclaved).
  2. Put the magnetic flea into the duran bottle in order to make it easier to mix the additives following autoclaving.
  3. Autoclave.
  4. Cool to 55°C.
  5. Place the media on a hot plate stirrer, to maintain add 1ml CaCl2, 1ml Cholesterol(5mg/ml i ethanol), 1ml MgSO4 and 25ml KH2PO4 buffer(1M).
  6. Mix well.
  7. Pipette into either 3cm (~3ml) or 6cm (~6ml) plates.

[-] RNAi Plates

  • NaCl - 3g
  • Peptone - 2.5g
  • Agar - 17g
  1. Dissolve the above chemicals in 975ml Milli-Q water. (Note: agar will not dissolve into solution until it has been autoclaved).
  2. Put the magnetic flea into the duran bottle in order to make it easier to mix the additives following autoclaving.
  3. Autoclave.
  4. Cool to 55°C
  5. Place the media on a hot plate stirrer, to maintain add 1ml CaCl2, 1ml Cholesterol(5mg/ml i ethanol), 1ml MgSO4, 25ml KH2PO4 buffer(1M), 0.5ml Carbenicillin(50mg/ml), 1ml IPTG(1M) and 1ml Nystatin(50 000U/ml)
  6. Mix well.
  7. Pipette into plates.

[-] RNAi Screening

Preparing for RNAi screen

Day One

  • Streak out RNAi clone from glycerol stock onto a flat plate. Leave overnight at 37°C.

Day Two

  • If the RNAi clone is from the library need to pick a single colony to restreak onto another flat plate, (this is your stock plate that lasts a month), make a plasmid prep, digest and run on a gel and finally sequence to verify the correct clone.
  • Once the RNAi clone has been sequenced verified, then need to pick a colony to grow in liquid LB + amp for ~6 hours (start growing at 9am). At around 4pm spot RNAi bacteria onto 6-well plates. Once the bacteria has dried, turn the plates over and leave on bench overnight.

NOTE: if spotting bacteria more than 4 days before putting the worms down need to put plates in the cold room. If plates have been put in the cold room take them out the night before you intend to put the worms down.

Performing RNAi screen

Day One

  • Bleach mothers that have started to lay eggs that have a eggs inside them.

Day Two

  • If feeding RNAi from L3, put hatched synchronised L1’s on OP50 plates, if feeding RNAi from L1 put straight onto RNAi plates.

Day Three

  • Check worms. If they look as though they are going to be older than late L3/ early L4 before the next day then put worms on RNAi plates. If they are still young wait until the next morning.

Day Four

  • Score 20 worms for age. If at L3/L4 then put worms on RNAi plates. Place RNAi plates at 20°C.

Day Five

  • If transferring worms, transfer worms from first well to middle wells. Check to make sure they have started laying eggs first.

Day Six

  • If transferring worms, transfer worms to the final well.

Day Seven

  • Nothing to do. If during the week can check plates.

Day Eight - Day Eleven

  • Score worms

[-] Single Worm PCR

  • Proteinase(at 20mg per ml)
  • NH4 Buffer(10x)
  • MgCl2(50mM)
  • dNTP’s(2mM)
  • Both Forward and Reverse Primer(10µM)
  • TAQ(usually Bio-Taq)
  1. Add proteinase K to 1x PCR buffer (85µl water, 10µl 10x PCR buffer + 5µl 20mg/ml proteinase K).
  2. Place 3µl 1x PCR buffer + proteinase K in the cap of a 200µl tube.
  3. Place a single worm in the drop of 1x PCR buffer + proteinase K.
  4. Place lid back on the tube and spin to the bottom of the tube in a microfuge at 14,000rpm for 15secs.
  5. Freeze tube at -80°C for 60 mins. (Can freeze for a number of days also). Freeze thaw worms 30 minutes before next step.
  6. To lyse the worm and release the genomic DNA, heat to 65°C for 90 mins. Inactivate proteinase K by heating to 95°C for 30 mins. NOTE: there is a PCR program to do both these steps.
  7. Perform PCR by adding 27µl of PCR master mix (1x NH4 buffer, 1.5mm MgCl2, 0.2mm dNTP’s, 0.5µm F and R primer and 0.1U/µl Taq polymerase.

Per reaction example:

  • Water - 16.6µl
  • NH4 buffer(10x) - 3µl
  • MgCl2(50Mm) - 0.9µl
  • dNTP’s(2Mm) - 3µl
  • Forward primer(10µM) - 1.5µl
  • Reverse primer(10µM) - 1.5µl
  • Taq - 0.5µ
  1. Run the PCR for 30-35 cycles.

[-] Embryo isolation using sucrose gradient

  1. Wash the worms off of each plate with M9 buffer into 15 ml tubes.
  2. Centrifuge for 1.25 minutes at 4000 rpm.
  3. Treat with Bleach Solution for between 5-7 minutes.
  4. Fill tube with M9 buffer, mix and centrifuge to pellet eggs and cell debris.
  5. Wash 2 times in quick succession with M9 buffer.
  6. To separate embryos from carcasses and cell debris, resuspend pellet in 10 ml of 30% sucrose solution (5 ml 60% sucrose plus 5 ml M9 buffer) and centrifuge for 1.25 minutes at 4000 rpm.
  7. Remove embryos (floating on top) with a transfer pipette along with no more than 5 ml of sucrose solution.
  8. Dilute to 15 ml with M9 buffer and centrifuge at 4000 for 1.25 minutes to pellet the embryos.
  9. Remove supernatant to leave synchronized eggs and hatch overnight in either M9 buffer or on plates without food.

[-] S-Media (for liquid RNAi) - 500mls

  • NaCl - 2.93g
  • K2HP04- 0.5g
  • KH2P04 - 3.0g
  • Cholesterol - 0.5ml
  • dH20 - 485.5ml

Autoclave then allow to cool down.

Once cool add the following sterile components:

  • 1M potassium citrate pH6 - 5ml
  • Trace Metal Solution - 5ml
  • 1M CaCl2 - 1.5ml
  • 1M MgS04 - 1.5ml
  • Nystatin (15mg/ml) - 0.5ml

Store at room temperature in tin foil.