Difference between revisions of "Team:Dundee/pmodal"

1. 5ml overnight cultures of the appropriate strains were set up.

2. The next day, 4x 995 µl of plain LB containing 1 µl ampicillin and 1 µl of chloramphenicol were inoculated with 5 µl of the overnight culture.

3. Three were induced with 0.5 mM, 1 mM and 2 mM of IPTG and one was left uninduced.

4. 200 µl of each culture was loaded onto a 96 well microtitre plate with 4 repeats.

5. The cultures were left to grow for 16 hours in the plate reader which took OD readings every 10 minutes.

1. 5ml overnight cultures of the appropriate strains were set up.

2. The next day, 4x 995 µl of plain LB containing 1 µl ampicillin and 1 µl of chloramphenicol were inoculated with 5 µl of the overnight culture.

3. 20 x 200 µl aliquots were loaded onto a 96 well microtitre plate.

4. After the OD₆₀₀=~0.6, three cultures were induced with 0.5 mM, 1 mM and 2 mM of IPTG and one was left uninduced (with 4 repeats).

5. The cultures were left to grow for 20 hours in the plate reader which took OD readings every 30 minutes.

1. A 150 ml overnight culture was set up for the desired protein.

2. 3 x 1 L of fresh LB containing 1 ml of appropriate antibiotics was inoculated with each 50 ml overnight culture and left to grow until an OD₆₀₀ =0.6-1 in a shaking incubator at 37^oC.

3. The cultures were subsequently induced with 1 mM IPTG and left to grow for 6 hours in a shaking incubator at 20^oC.

4. They were then centrifuged for 20 minutes at 4200 rpm at 4^oC.

5. The pellets were then resuspended in 5 ml of ice cold 50 mM 7.5 Tris and transferred to a 50 ml falcon tube and then topped up to 40 ml with 50 mM 7.5 Tris.

6. They were then spun down again at 4000 rpm for 15 minutes at 4^oC, the supernatant discarded and pellets stored at -20^oC.

7. Once purification could proceed, the pellets were thawed on ice and resuspended in 40 ml of 50 mM Tris containing DNAase, 1 x protease inhibitor tablet and lysozyme. (Note: If purifying a membrane protein, 1% DDM and 5 M urea was also added).

8. The cells were lysed using a cell disruptor.

9. Afterwards the lysate was spun down in a J25.50 rotor at 15000 rpm at 4^oC for 40 minutes.

10. The supernatant was removed and transferred to a 50 ml falcon tube. The pellet was kept for testing. Both were kept on ice.

11. Using FPLC, the supernatant was loaded onto a Ni²⁺ column.

12. The protein was eluted using an increasing concentration of imidazole. The two buffers used were Buffer A: 50 mM Tris, 200 mM NaCl, 20 mM imidazole and Buffer B: 50 mM Tris, 200 mM NaCl, 1 M Imidazole. (Note: If purifying a membrane protein 0.02% DDM was added to these buffers.)

13. The fractions corresponding to any peaks were retained and concentrated using an appropriate filter size. A small amount of each fraction could be ran on an SDS gel at this stage.

14. Using FPLC, the sample was then loaded onto a SEC column and any fractions corresponding to a peak were ran on an SDS gel to check for the presence of the desired protein.

15. Fractions containing the desired protein were collated, to which 20% glycerol was added.

16. The samples were then flash frozen and stored at -80^oC.

1. Take 3 independent colonies from each plate of your controls and samples and place each colony into 5 ml of LB with the appropriate antibiotics.
2. Leave to grow overnight at 30°C.
3. Take 50 μl of each overnight and inoculate 5 ml of fresh LB containing the appropriate antibiotics. Do this twice for every independent colony.
4. Allow these to grow at 30°C shaker until the O.D of each inoculate is between 0.3-0.5
5. Once the O.D is between 0.3-0.5 take 3 x 1 ml samples of each culture and place into separate Eppendorf tubes.
6. Take a further 1 ml and place into a cuvette and subsequently measure the OD₆₀₀ marking down the OD for each replica.
7. Spin down the 1 ml cultures of each replica at 13,000 rpm for 3 minutes.
8. Remove the supernatant from each 1 ml culture and freeze cell pellet for later use in the β-galactosidase Assay.

1. Resuspend cell pellet in 800 μl of buffer 2 (100 mM dibasic sodium phosphate (Na2HPO4); 20 mM KCl ; 2 mM MgSO4 ;0.8 mg/mL CTAB (hexadecyltrimethylammonium bromide) ; 0.4 mg/mL sodiumdeoxycholate) and subsequntly add 5.4 μL/mL beta-mercaptoethanol.
2. Add 50 μl of SDS (0.1%) to each sample.
3. Add 50 μl of Chloroform to each sample and vortex for 30 seconds.
4. Make substrate solution (60 mM Na2HPO4; 40 mM NaH2PO4; 1 mg/mL o-nitrophenyl-β-D-Galactoside (ONPG); 2.7 μL/mL β-mercaptoethanol)
5. Add 200 μl of substrate solution to each sample marking down the time it was added in relation to the first sample.
6. Wait for an observable color change and mark down the time that the color change occurred.
7. Add 600 μl of NaCO3(1M) once the color change has occurred.
8. Spin down the samples at 13,000 rpm for 3 minutes and place the supernatant into a cuvette.
9. Measure the OD₄₂₀.

1. A 5 ml overnight culture of the desired strain was grown at 37 ^oC.
2. Equalise OD₆₀₀ if required. Dilute culture 1:1000.
3. Aliquot 198 µl into the required amount of wells in a 96-well plate.
4. Add additional substances and controls as required.
5. Preheat platereader to required temperature and start experiment.
6. Retrieve and analyse data.

1. A 5 ml overnight culture of the desired strain was grown at 37^oC.
2. Subsample ¹/₁₀ of the volume of the subculture. I.e. 1 ml of the overnight culture into 10 ml fresh LB.
3. Grow subculture at at 37 ^oC for 2 h.
4. Spin down subculture at 4000 rpm at 4^oC for 10 min.
5. Resuspend the pellet in ice cold transformation buffer. Use ¹/₁₀ of the initial volume of the subculture.
6. Use 100 µl per transformation.

1. Agar plates with required antibiotic were taken out of 4^oC to warm to room temperature.
2. Cells or colonies were picked up from the source with a toothpick under sterile conditions.
3. Fresh agar plates were inoculated with cells on the toothpick.
4. Agar plates were incubated at 4^oC over night.

For sequencing a sample, the following ingredients were added to a microcentrifuge tube:

• Volume of sample equivalent to 550ng DNA
• 2 µl of 3 mM primer
• Remaining volume of water for a total volume of 30 µl

The samples were submitted to DNA sequencing and services.

1. Agar plates with required antibiotic were taken out of 4^oC to warm to room temperature.
2. Cells or colonies were picked up from the source with a toothpick under sterile conditions.
3. 5 ml of LB medium, supplemented with the required antibiotic, were inoculated with the toothpick.
4. Tubes were incubated at 37^oC in a rotary incubator at 200rpm over night.

1. The desired DNA fragment was excised from the agarose gel with a clean, sharp scalpel.
2. The gel slice was transferred into a microcentrifuge tube. 700 µl of Buffer QG was added.
3. The gel slice was incubated in a water bath at 50°C for 10 min (or until the gel slice has completely dissolved). The tube was vortexed every 2–3 min to help dissolve gel.
4. After the gel slice had been dissolved completely, the color of the mixture was checked that it was yellow (similar to Buffer QG without dissolved agarose). If the color of the mixture was orange or violet, 10 μl 3 M sodium acetate, pH 5.0 would be added, and mixed. The color of the mixture would turn yellow.
5. 230 µl of isopropanol was added to the sample and mixed.
6. A QIAquick spin column was placed in a 2 ml collection tube. The sample was applied to the QIAquick spin column and centrifuged for 1 minute. The flow-through was discarded.
7. 500 µl of QG buffer was added to the QIAquick spin column and centrifuged for 1 minute. The flow-through was discarded.
8. 750 µl of Buffer PE was added to the QIAquick spin column and centrifuged for 1 minute. The flow-through was discarded.
9. The QIAprep spin column was spun for an additional minute to remove residual wash buffer.
10. The QIAprep spin column was placed in a clean 1.5 ml microcentrifuge and the DNA eluted by adding 30 µl of H2O to the QIAprep spin column and centrifuging for 1 minute.