Lab techniques

Question 1

Cell tissue culture - passaging adherent cells

Answer 1

1. Set up tissue culture hood, sterilise using ethanol and place all needed supplies in it
2. Examine cells for contamination under the microscope
3. Remove media using a macroman fitted with a sterile 10 ml pipette tip
4. Wash cells with 10 ml of PBS, using a new 10 ml pipette tip, then discard the PBS
5. Add 2 ml of trypsin EDTA to lift the cells and incubate for 10 minutes
6. Check under the microscope that the cells have detached, tap the flask gently if necessary
7. Using a 5 ml pipette tip, break up the clumps of cells by pipetting up and down
8. Add 8 ml of media (e.g. DMEM) to the cells
9. Take a 2 ml sample and transfer into a ladled LP3 tube
10. Using a haemocytometer and counter, determine the cell density under a microscope
11. Based on you cell count determine the ratio the cells need to be passaged
12. Remove the excess cell solution and top up with media to make up 10 ml in the flask (or 20 ml in the large flasks)
13. Update the passage number on the flask

Question 2

Trypan blue

Answer 2

1. Set up tissue culture hood, sterilise using ethanol and place all needed supplies in it.
2. Lift cells and seed 12 well plates, at a density of 3x104 cells/cm2, allow cells to attach overnight.
3. Treat cells with the following drugs alone and/or in combination: gemcitabine (100µM), tumour necrosis factor (TNF)-related apoptosis-inducing ligand (100ng/µl), chloroquine (50 µM) and MG132 (30µM).
4. Following treatment, collected the media for each treatment into label Eppendorf tubes. Spin down and discard the media.
5. Rinse wells with 300µl PBS and add to the appropriate Eppendorf tube.
6. Lift remaining cells from wells using 400µL Accutase, incubate 10 to 15 minutes and add the lifted cells to the Eppendorf’s as well.
7. Transfer 200 µl of cell solution to a new labelled Eppendorf and add 200 µl of Trypan blue (a 1:1 ratio). Mix by pipetting up and down.
8. Using a haemocytometer and counter, count the number of viable (white cells) and dead cells (blue cells).

Question 3

MTT Assay

Answer 3

1. Set up tissue culture hood, sterilise using ethanol and place all needed supplies in it.
2. Lift cells and seed 96 well plates, at a density of 3x104 cells/cm2, allow cells to attach overnight. Add media to the remaining wells and 10 ml of media between the wells. (see figure below)
3. Treat cells with the following drugs alone and/or in combination: gemcitabine (100µM), tumour necrosis factor (TNF)-related apoptosis-inducing ligand (100ng/µl), chloroquine (50 µM) and MG132 (30µM).
4. Following treatment add 25 µl of 0.5mg/ml MTT to the wells C to F from columns 1 to 12. Incubate for 4 hours.
5. Solubilise formazan crystals using 100 µl of 10% SDS and incubate
   overnight.
6. Read absorbance at 595 nm.

Question 4

Western blotting: Protein isolation from whole cell extract

Answer 4

1. Seed 60 cm2 dishes at a density of 3x104 cells/cm2, allow cells to attach overnight.
2. Treat cells with the following drugs alone and/or in combination: gemcitabine (100µM), tumour necrosis factor (TNF)-related apoptosis- inducing ligand (100ng/µl), chloroquine (50 µM) and MG132 (30µM).
3. Following treatment, transfer media into a chilled labelled 50 ml falcon tube. This media may contain cells that had responded to the treatment and were no longer adherent.
4. Place dishes on ice and harvest attached cells by mechanically scraping into 7 ml PBS. Add into the same chilled labelled 50 ml falcon tube. Repeat with another 5 ml PBS.
5. Centrifuge at 10,000g at 4 °C for 10 minutes. Discard supernatant.
6. Re-suspend the pellet in 1 ml of PBS and transfer into labelled Eppendorf.
7. Spin down at 11,000rpm for 10 seconds. Discard supernatant.
8. Re-suspend cells in 35 µl lysis buffer, pipette up and down to break up pellet.
9. Vortex 5 seconds and incubate on ice for 5 minutes.
10. Following incubation, sonicate 3 times to break open cells
11. Spin down in a chill spinner at 14000 g for 10 minutes at 4 °C
12. Transfer supernatant into new labelled Eppendorf tube.

Question 5

Western blotting:

Sodium Dodecyl Sulphate (SDS) Polyacrylamide Gel Electrophoresis

Answer 5

1. To resolve protein within the cell extracts, SDS Polyacrylamide Gel Electrophoresis (PAGE) has to be performed.
2. Prepare the SDS polyacrylamide gel. Pour the resolving gel first followed b the stacking gel.
3. Using the concentrations of samples determined using the Bradford assay, prepare 5 µg of protein samples in 2x SDS samples buffer.
4. Boil for 5 minutes at 95°C to denature the protein.
5. Load gel with 10 µl of samples into wells, 4 µl biotinylated marker and 5 µl rainbow marker.
6. Run the gel at 175 volts, until the bromophenol blue in the sample
   buffer has run off the gel

Question 6

Western blotting:

Semi-dry transfer

Answer 6

1. Dismantle the gel apperartus and equilibrate the gel in Towbins transfer buffer.
2. pre-soak PVDF membrane in methanol and then transfer into Towbins transfer buffer.
3. Onto the anode of the semi dry transfer apparatus, place three layers of Whatman filter paper soaked in Towbins transfer buffer followed by the PVDF membrane.
4. Carefully place the gel ontop followed by additional place three layers of Whatman filter paper soaked in Towbins transfer buffer.
5. Run the apparatus at 15 volts for 50 minutes
6. Fix the protein onto the PVDF by incubating with Glutaraldehyde (0.2% v/v in PBS) for 10 minutes at room temperature.
7. Next transfer the membrane was into blocking buffer and incubate at room temperature for 1 hour on an orbital shaker.

Question 7

Western blotting:

Chemiluminescence

Answer 7

1. Wash the membrane in 10 ml TBS-Tween buffer for 2 minutes. Repeat.
2. Incubate with a primary antibody in TBS-Tween overnight at 4°C on an orbital shaker.
3. Discard the primary antibody and wash with 10 ml TBS-Tween for 2
   minutes. Repeat 2 times.
4. Incubate the blot on an orbital shaker for 1 hour at room temperature with the appropriate secondary antibody ( 5 µl anti mouse-HRP-conjugated antibody solution and anti-biotin anti-body.
5. Discard the secondary antibody and was the membrane with TBS- Tween a further 3 times.
6. To develop the blots, take them to the dark room. Incubate the membrane with 5ml of 1x LumiGLO reagent for 1 minute.
7. Following the incubation place the membrane in an X-ray cassette between two sheets of overlay plastic. In the dark place an X-ray film on top and expose for 5 seconds.
8. Develop the film in Konica SRX-101A. Repeat by exposing the X-ray film to the membrane for varying amount of time.
9. Loading can be assessed by probing the blot for an anti- housekeeping gene such as GAPDH

Question 8

PCR:

Setting up PCR

Answer 8

1. Set up PCR machine to denaturing at 95 °C (20 sec), annealing at 59°C (20 sec) followed by amplification at 68 °C (for 1 min) and repeat for 30 cycles.
2. In PCR tubes combine and start PCR.
   2 µl cDNA
   0.5 µl forward primer
   0.5 µl reverse primer
3. 5 µl mastermix – containing dNTPs, Taq polymerase and buffer Up to 25 µl water

Total volume: 25 µl

Question 9

PCR:

Visualising results on agarose gel

Answer 9

1. Prepare agarose gel:
   In microwave safe container combine TAE buffer with 1.2 % agarose Microwave
   Allow to cool and pour into tray Allow to set
2. Load samples into each well and run gel
3. Image your gel and analyse results

Question 10

PCR:

DNA purification

Answer 10

1. Use the NEB PCR &DNA clean up kit. Begin with adding binding buffer to your PCR sample at a ratio of 5:1 (e.g. to a reaction of 20 µl add 100 µl buffer) and mix well by pipetting up and down. Do not vortex.
2. Insert a column (provided in the kit) on to a new tube and transfer the sample into a column.
3. Spin for 1 minute at 13,000rpm (the DNA will now be bound to the column). Remove column and discard flow through.
4. Re insert column into the tube and add 200 µl DNA wash buffer. Spin for another 1 minute at 13,000rpm. Then repeat with another 200µl of DNA washing buffer.
5. Transfer column to a clean tube and add 20 µl of elusion buffer to
   the centre of the matrix. Spin for 1 minute.
6. The elute now contains your DNA sample.

Question 11

Cloning DNA

Answer 11

1. PCR gene of interest from cDNA
2. Ligate into pGEM-T Easy, by seting up a ligation buffer. Mix the reaction and incubate for 1 hour at room temperature.
3. Transform into JM109 (E coli).
4. Agar plates contain Ampicillin (10g/ml) and spread with IPTG/X-gal.

Question 12

Bacterial transformation

Answer 12

1. Thaw 20µl aliquots of bacteria on ice. The bacteria are very fragile and should not be vortexed, or pipetted vigourously.
2. Gently add DNA for transformation while inserting the pipette tip into the bacterial suspension. Leave on ice for 30 minutes. Add no more than 10µl of DNA. For ligations, I usually add 1µl of a 10µl reaction. For transformation from a plasmid, add about 50ng. Place agar plates in the incubator to allow them to warm up.
3. Heat shock bacteria at 42°C for EXACTLY 45 seconds and return to ice for 2 minutes.
4. Add 80µl of SOC (or LB - But this reduces efficiency) and place in 37°C water bath for 1 hour.
5. Plate onto LB agar (or whatever) with appropriate antibiotic selection. For blue/white selection, agar plates should be spread with 20µl of 50mg/ml X-gal and 100µl of 100mM IPTG, then allowed to absorb for at least 30 minutes at 37°C before plating bacteria. Do this at step 2.
6. Incubate plates for several hours to overnight at 37°C and pick colonies

Question 13

DNA Extraction from bacteria: Mini prep

Answer 13

1. Use the NEB Plasmid Miniprep Kit.
2. Pellet 1.5 ml bacterial culture by centrifugation for 30 seconds. Discard supernatant.
3. Resuspend pellet in 200 μl Plasmid Resuspension Buffer. Vortex or pipet to ensure cells are completely resuspended. There should be no visible clumps.
4. Add 200 μl Plasmid Lysis Buffer, gently invert tube 5–6 times, and incubate at room temperature for 1 minute. Colour should change to dark pink, and solution will become transparent and viscous.
5. Add 400 μl of Plasmid Neutralization Buffer, gently invert tube until neutralized, and incubate at room temperature for 1 minute. Sample is neutralized when colour is uniformly yellow and precipitate forms.
6. Centrifuge lysate for 2–5 minutes. For best results, and especially culture volumes > 1 ml, we recommend a 5 minute spin to ensure efficient RNA removal by RNase A. Pellet should be compact; spin longer if needed.
7. Carefully transfer supernatant to the spin column and centrifuge for 1 minute. Discard flow-through.
8. Re-insert column in the collection tube and add 200 μl of Plasmid Wash Buffer
9. Centrifuge for 1 minute. Discarding the flow-through is optional.
10. Add 400 μl of Plasmid Wash Buffer 2 and centrifuge for 1 minute.
11. Transfer column to a clean 1.5 ml microfuge tube. Use care to ensure that the tip of the column does not come into contact with the flow-through. If there is any doubt, re-spin the column for 1 minute.
12. Add ≥ 30 μl DNA Elution Buffer to the center of the matrix. Wait for 1 minute, then spin for 1 minute to elute DNA. Nuclease-free water (pH 7–8.5) can also be used to elute the DNA. Yield may slightly increase if a larger volume of DNA Elution Buffer is used, but the DNA will be less concentrated. For larger size DNA, (≥ 10 kb), heating the elution buffer to 50°C prior to use can improve yield.

Question 14

Transfection (complex)

Answer 14

1. Seed cells at a density of 1x 105 cells in 500uL in 4 well plates
2. 1.5mL tube, dilute 1.6ul-2uL Lipofectamine in 50 μl of Opti-MEM® in serum free medium (ratio 1:2). Incubate for 5 minutes at room temperature. (Note: non-transfected control won’t contain Lipofectamine so just add 50uL of medium to the tube).
3. 1.5mL tube, dilute DNA (0.8 ug) in 50 μl of Opti-MEM® I Reduced Serum Medium without serum (or other medium without serum). Mix gently. (Note: non-transfected
   cells and vehicle controls won’t contain DNA
   so add only 50uL of medium to the tube).
4. Combine the diluted DNA with diluted
   Lipofectamine™ 2000 (total volume = 100 μl). Mix gently and incubate for 20 minutes at room temperature (solution may appear cloudy). Note: Complexes are stable for 6 hours at room temperature.
5. Add the 100 μl of complexes to each well containing cells and medium. Mix gently by rocking the plate back and forth.
6. Incubate cells at 37°C in a CO2 incubator for 18-48 hours prior to testing for transgene expression. It is not necessary to change the medium, but medium may be replaced after 4-6 hours. 5. For stable cell lines: Passage cells at a 1:10 (or higher dilution) into fresh growth medium 24 hours after transfection. Add selective medium (if desired) the following day.

Question 15

Transfection (simplified)

Answer 15

1. Seed cells to be 70-90% confluent at transfcetion
2. Dilute four amounts of Lipfectamine reagent in opti-MEM medium
3. Dilute DNA in Opti-MEM medium
4. Add diluted DNA to diluted Lipofectamine 2000 reagent (1:1 ratio)
5. Incubate
6. Add DNA-lipid complex to cells
7. Visualise/analyze transfected cells

Question 16

Zebrafish staining: Phalloidin and sytox-orange

Answer 16

1. Fix embryos over-night at 4 ºC in Paraformaldehyde 4% in PBS
2. Wash twice with PBS + Triton 0.3% (10min each)
3. Incubate with Phalloidin 1:50 (stock at 24uM) and Sytox 1:10000 (stock at 5mM) in PBS + Triton 1% + DMSO 1%, at RT for at least 2hours
4. Rinse twice in PBS + Triton 0.3%
5. Mount in low-melt point agarose 1% in a glass bottom petri dish and cover with PBS

Question 17

Riboprobe preparation

Answer 17

1. Prepare transcription reaction

10X transcription reaction buffer 2µl
DTT	2µl
10X DIG-NTPs mix 2µl
Rnase-inhibitor	1µl
T3 Polymerase	1µl
PCR DNA product 12µl

2. Incubate for 2h at 37°C.
3. Incubate for 10 minutes at 37°C, with 1 µl of DNAse (RQ1).
4. Precipitate by adding:

80 µl Water
10 µl 4M LiCl
300 µl Ethanol

5. Incubate overnight at -20 ˚C.
6. Centrifuge at 4°C for 20min at maximum speed
7. Do not let dry very much, and resuspend straightaway in 25ul of Hyb+

Question 18

Zebra fish: In situ hybridisation

Answer 18

DAY1
1. Fix embryos by incubating overnight in 500µl 4% PFA at 4˚C.
2. Wash with 500µl PBS.
3. Incubate with 500µl Methanol at -20˚C at least overnight (samples can
be stored at this point for as long as needed).

DAY2
4. Wash 2 times with 500µl PBS triton 0.1% (0.1% PBST)
5. Proteinase K treatment: only for embryos 24hpf and older PK stock at 10mg/ml
6. Wash gently twice with PBST
7. Refix with PFA 4% 20min at Room Temperature
8. wash 5 times 5min in PBST
9. Rinse in Hyb+/PBST 1:1
Hyb+: 5XSSC, 50%formamide, 0.1%triton, 5mg/ml torula RNA, 50ug/ml heparin
SSC 20X: NaCl 3M; NaCitrate 0.3M
10. Prehybridise in Hyb+ for at least 1h at 68°C
samples can be stored at -20˚C for some time
11. Replace with diluted probe (1:300 for strong probe, 1:100 unknown strength)
12. Incubate over night at 68°C

DAY3
13. Recover probe
14. Wash 4 times 20min with Hyb-
Hyb-: 5XSSC, 50%formamide,0.1%triton (at 68°C)
15. Rinse once with 2XSSC (at 68°C)
16. Wash twice 20min with 0.2XSSC (at 68°C)
17. Wash 4 times 15min with PBST (back at room temperature)
18. Block for at least 1h with MABlock (0.1M maleic acid pH7.8+5%BSA from Roche)
19. Incubate with anti-DIG antibody (Roche) 1:6000 overnight at 4°C
Embryos can stay in step 19 for several days

 DAY 4
20.	Wash 4 times 15min with PBST
21.	Prepare staining buffer:
0.1M	  Tris HCL pH9.5 (keep 1M stock)
50mM	MgCl (keep 1M stock)
0.1M  	NaCl (keep 4M stock)
0.1%	 triton (keep 10%stock)
22.	Wash 3 times 5min in staining buffer
23.	Prepare staining solution:
1µl	NBT
3.5µl	BCIP
1ml	staining buffer

24. Incubate in the dark, without agitation, check every once in a while.
25. Stop reaction washing a few times with PBST
26. Refix with PFA 4% overnight at 4°C

DAY 5
27. Wash in PBS, 30%, 50% and 70% glycerol. Store in 70% glycerol.

Question 19

Zebra fish drug screen

Answer 19

1. Take the LOPAC1280 library at a concentration of 10mM and make a working stock at 1 mM (1/10, 1µl of drug in 20µl fish water).
2. Select embryos to use at 4-6hpf.
3. In a 96-well plate add 3 embryos per well in 80µl fish water.
4. Add 20µl of fish water with the drug so that the final concentration is 10µM and incubate for 24/48 hours.
5. Screen embryos for phenotype at 24, 48 and 72 hpf.
6. After the initial screen do one of the following:
   ◦ Determine window of time in which the drug works by doing treatments at different stages.
   ◦ Do more detailed analysis by in situ.
   ◦ Analyse the effect in a particular system/ tissue by doing the treatments
   in a transgenic line.