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Team:St Andrews/Lab-book
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Latest revision as of 02:56, 27 September 2012
Lab book
Protocols
1. Frozen cells were thawed from -80 °C.
2. Thawed cells (10 μL) were mixed with vector (1 μL), and left on ice for 30 min.
3. LB liquid medium (1 mL) was warmed at 37°C for 30 min.
4. The cell/vector mix was heated at 42 °C for 45 seconds.
5. Warm LB (50 μL) was added to the cell vector mix.
6. Mixture was shaken at 37 °C for 30 min.
7. Mixture was plated onto ampicillin agar plates and left overnight.
8. Recombinant E. Coli was harvested from agar plate by scratching gently with the scraper.
9. The scraper tip was placed into Falcon tubes with LB (10 mL) and ampicillin (1 μL) to transfer the E. Coli into medium.
10. The E. Coli was incubated in a shaker at 37 °C for less than 20 hr.
1. Preparing 1% agarose solution: Agarose powder (1 g) was dissolved in TAE (Tris base, acetic acid and EDTA) buffer (100 mL). This was heated in the microwave until the solution was transparent. Ethidium Bromide (8 μL) was added.
2. The solution was poured into an assembled gel rigand allowed to set.
3. Prepared samples were loaded into the wells(5-20 μL) along with DNA ladder (5 μL).
4. The gel was run at 80 V for 20-50 min.
5. The gel was visualised under UV light
Ratios for sample preparation vary depending on the type of sample:
- Miniprep - use 4 μL sample and 2 μL dye
- Digestion - use 1 μL sample and 2 μL dye
- PCR- use 10μl sample and 1μl dye
When using GoTaq®, simply load 10 μL onto the gel, as dye is already included.
Always make up to 10 μL using water.1. The sample (30μl) was prepared in the following sequnce:
2. The sample was incubated at 37 °C in a water bath. Timings depended on the restriction enzymes brand.
1. Prepare 20 μM primer working stock for both forward and reverse primers
2. Prepare PCR according to polymerase brand:
High-fidelity:
Clontech
KOD
GoTaq
1. PCR tubes were prepared in the following sequence:
to make a 50μL PCR solution
2. An Eppendorf tube was prepared for each PRC tube.
3. Using a small pipette tip, one colony was scratched off a plate and dipped into the tip into the PCR tube. Tips were then stored in the corresponding Eppendorf tubes (for further incubation, if positive).
4. The PCR was ran using optimal annealing temperatures for the required gene, and then the samples of the PCR run straight on electrophoresis.
6. Positive results from the samples were incubated from the corresponding tips.
1. Add phosphatase buffer to sample in accordance with its concentration factor.
2. Add 1μL phosphatase.
3. Incubate 60 mins at 37°C.
4. Heat-shock for 5 mins at 65°C.
Ligation molar ratio calculation:
Exposure intensity from UV light was used to quantify the DNA in ng (i.e. the concentration of both insert and vector) against ladder position for sequence length (i.e. molar weight ratio of the two)
Rule of 3: Three times as much insert as vector ensures successful ligation results
1. Ligation solution (20 μL) was prepared
- 10x buffer (2 μL)
- T4 ligase (2 μL)
- Vectors and insert (~17 μL), according to calculated ratio
- make up to a total of 20 μL using water
2. The ligation mixture was allowed to stand at room temperature for 10-30 minutes..
3. The ligase-treated vector-insert mixture was mixed with E. Coli (DH5-α)(60 μL), then the standard transformation protocol was followed with shaking for 1 hr; finally LB (200 μL) was added.
p>1. The optimal density (OD) of a portion of the sample (1 mL) was measured in the spectrophotometer using LB as a blank.
2. If the OD was approximately 0.5 the sample was induced. The sample could be incubated further or diluted if necessary.
2. IPTG (1 mM) was added.
1. The E. Coli was spun down in a centrifuge.
2. The supernatant was discarded and phosphate buffer saline(PBS, 1 mL) was added to resuspend the pellet before the mix was transferred to an Eppendorf tube.
3. The E. Coli was spun at full speed for 1 min., the supernatant discarded and the pellet was put on ice for storage.
4. Lysozyme solution (10 mg/mL) was prepared in lysis buffer.
5. Lysozyme solution (200 μL) was added to each pellet and they were resuspended and vortexed before being incubated in a shaker (37 °C) for 30 min. to 1 h.
6. The cells were sonicated.
7. Total sample (10 μL) was removed and 2x dye (10 μL) was added.
8. \the original tubes were spun for 1 min. at full speed and the supernatant was removed, the mass was resuspended with PBS (200 μL).
9. Membrane sample (10 μL) was taken from the resuspended solution and 2x dye (10 μL) was added.
10. The protein samples were denatures on a heating block (95° C or 75°C for membrane protein).
11. The samples were cooled on ice for 1 min., then loaded on to the protein gel (prepared according to SDS-PAGE standard protocol) and run at 150 V for around 45 min.
1. Making the membrane transfer sandwiches: 2x A2 at the bottom, then 2x A2.
2. The membrane, GE Healthcare HyBond ECL films, was cut out using a glass template; the membrane was put onto moist A2 filter papers and pressed flat using a roll.
3. The gel slides were opened using a wedge; removed the thinner slide and, using the wedge, prized the protein gel from the thicker plate and onto the membrane.
4. The airbubbles were gently pressed out between the membrane and the gel.
5. CAT filter paper was added.
6. The power pack was run for 1hr (40 mA).
7. If the restained markers have transferred, place the CAT filter papers onto a rocker and add stain. If using Ponceau, the stain can be reused.
8. Destain was done by rinsing with water.
21. Milk powder (50 mL 5%) was added and left to rock for 2 hours at room temperature. If leaving overnight, rock in cold room using lower speed.
9. The blocking milk solution was decanted, and replaced with milk (10 mL 1%) with antibodies (5μl of diluted). This was rocked for 1 h.
10. The milk-antibody solution was poured off and washed with PBS (0.3%) and Tween, three times at 10 min intervals.
11. A solution of "Supersignal west Fermto trail" kit (1:1 ratio, 1 mL per membrane).
12. The membrane was made moist with the visualization solution, covered for 5 min, then the excess liquid was removed.
13. The membranes were developed in a dark room.
Project-specific procedures
ω3-Synthesis
1. The E. Coli was spun down in a centrifuge(800g, 10min).
2. With the supernatant discarded, PBS (1 mL) was used to resuspend the pellet, then transferred to Eppendorf tubes.
3. The E. Coli was spun again at full speed for 1 min. and the supernatant discarded.
4. The pellet was resuspended using PBS (100 μL) and then transferred to glass sample vials.
5. An organic mixture of chloroform:methanol (1:2(v/v), 375 μL) was added for lysis.
6. The sample was vortexed for 1 h in the cold room, with chloroform (125 μL) and water (125 μL) added to allow phase separation (lower organic layer contains lipids); and left to stand for at least 10 min.
7. The sample vials were spun at 1000g at room temperature for 5 min.
8. The lower organic layer was transfered to a new glass vial avoiding the upper aqueous layer at all costs.
9. The resultant lower phase lipid extract was then dried under N2, and the fatty acid stored in the fridge.
9. The samples can be re-dissolved in chloroform:methanol (1:2) and are ready for Electrospray-mass spectrometry analysis for phospholipids and can be further processed for fatty acid derivation and analysis by Gas Chromatography-Mass Spectrometry.
1. Per cell type, there will be 3 samples:
- A (negative control)
- B (no added substrate beyond induced 18:1)
- C (addition of substrate PC lipid 18:1)
2. After labelling all screw-cap Eppendorfs, add the following assay components to each B and C tube:
- tricine buffer (20 μL, 40 μM), made up to pH 8 with KOH
- MgCl2 (20 μL of 10 μM)
- Ferredoxin (10 μL)
- NADPH (10 μL, 5μM)
- Catalase (30 μL)
- NADPH+ (10 μL)
3. The membrane sample (100 μL), prepare d as in the lipid extraction, was added to the mixture. Lysozyme (500 μL) was added, and resuspended in tricine buffer. The Bradford assay was run to determine protein content.
4. The sample was vortexed at 4°C for 2 min.
5. It was put in a 37°C water bath.
6. Chloroform (250 μL) and water (250 μL) inhibited the assay reaction and allowed for phase separation.
7. Samples were used to carry out mass spec analysis.
1. Aliquots of the lipid extract were transferred to 2mL glass vessels and dried under nitrogen.
2. Base hydrolysis to release fatty acids was done using of concentrated ammonia (500 µL) and 50% propan-1-ol (50%) 1:1, followed by incubation for 5 h at 50 °C.
3. After cooling, the samples were evaporated to dryness with nitrogen and were dried twice more from methanol : water (1:1) (200 µL) to remove all traces of ammonia.
4. The protonated fatty acids are then extracted by partitioning between HCl (500 µL, 20 mM) and of ether (500 µL); the aqueous phase was re-extracted with fresh ether (500µL) and the combined ether phases were dried under nitrogen in a glass tube.
5. The fatty acids were converted to fatty acid methyl esters (FAMEs), by adding diazomethane (3x 20 µL aliquots) to the dried residue, while on ice. After 30min, the samples were allowed to warm to RT and left to evaporate to dryness in a fume hood.
6. The FAME products were dissolved in dichloromethane (10-20 µL) and analysed (1-2 µL) by GC-MS on a Agilent Technologies (GC-6890N, MS detector-5973) with a ZB-5 column (30M x 25mm x 25mm, Phenomenex), with a temperature program of at 70 °C for 10min followed by a gradient to 220 °C at 5 °C /min and held at 220 °C for a further 15min
7. Mass spectra were acquired from 50-500 amu. The identity of FAMEs was carried out by comparison of the retention time and fragmentation pattern with a Bacterial FAME standard (Supelco).
The Bradford protein assay was carried out in accordance with standard procedure as described in BRADFORD, M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem, 7;72:248-54.
Metal-binding peptides
Primer annealing was carried out to create short peptides.
10 μL of 200 μM of each primer was put into the PCR machine.
PCR Programme:
1. The E. coli was spun down in a centrifuge.
2. The supernatant was discarded and phosphate buffer saline (PBS, 1 mL) was added to re-suspend the pellet. The mixture was transferred to eppendorf tubes.
3. The E. coli was spun down at full speed for 1 min. The supernatant was discarded and then put on ice for storage.
4. Lysozyme solution (10 mg/mL) was prepared in lysis buffer.
5. Lysozyme solution (200 μL) was added to each pellet and re-suspended. Benzonase nuclease (1 µL) was added and 1 protease inhibitor cocktail pill was disolved in the solution; this was vortexed and incubated in a shaker at 37 °C for 30 min. to 1 h.
6. The cells were sonicated.
7. The sonicated cells were spun down at full speed for 1 min in a centrifuge.
8. The Nickel beads were prepared. Ni-NTA slurry per sample(100 µL). This was spun in centrifuge at full speed, and the supernatant removed . PBS was added (100 µL), the pellet was re-suspended and spun down at full speed for 1 min.
9. The supernatant was removed; PBS was added (100 µL), the pellet was re-suspended and transferred to eppendorf tubes (100 µL). This was spun down at full speed for 1 minute in a centrifuge,and the supernatant was removed. The supernatant of the sonicated cells was added and the mix was re suspended.
10. The samples were agitated for 30 min to 3 h in the cold room. The samples were spun down at full speed for 1 minute in centrifuge and the supernatant was removed.
11. NINTA elution buffer (100 µL) was added and samples were agitated for 10 to 30 min. in the cold room.
12. The samples were spun down at full speed for 1 min. and 10 µL of supernatant was removed.2x dye was added to the supernatant (10 µL).
13. The protein samples were denatures on the heating block at 95° C.
14. The samples were cooled on ice for 1 min., and then loaded to the gel (prepared according to SDS-PAGE standard protocol) and run at 150 V for 45 min.
1. The E. coli were spun down in a centrifuge.
2. The supernatant was discarded and phosphate buffer saline (PBS) (1 mL) was added to re-suspend the pellet. The mixture was transferred to eppendorf tubes.
3. The E. coli in the tubes were spun down at full speed for 1 min. The supernatant was then discarded and stored on ice.
4. The lysozyme solution (10 mg/mL) was prepared in lysis buffer.
5. The lysozyme solution (200 μL) was added to each pellet and re-suspended. Benzonase nuclease (1 μL) was added, and one protease inhibitor cocktail pill was dissolved in the mixture. This was then vortexed and incubated in the shaker (37 °C for 30 min to 1 h).
6. The cells were then sonicated.
7. The sonicated cells were then spun down at full speed for 1 min in the centrifuge .
8. GST beads were prepared. GST slurry (100 µL) was added to each sample and spun down at full speed. The supernatant was then removed. PBS (100 µL) was added to each sample, and the mixture re- suspended and spun down at full speed for 1 min.
9. The supernatant was then removed and PBS (100 µL) added per sample, re-suspended and transferred to eppendorf tubes, 100 µL per sample. The mixture spun down at full speed for 1 min and supernatant was then removed. The supernatant of the sonicated cells was then added.
10. 10. The samples were agitated for 30 min to 3 h in the cold room. Samples were spun down at full speed for 1 min. Supernatant was removed.
11. GST beads were washed with PBS, 200 µL per sample. Samples were spun down at full speed and supernatant was removed. Glutathione solution (50 µl, 100 mM) was added . The samples were agitated for 10 to 30 min in the cold room.
12. The samples were spun at full speed for 1 min. 10 µL of supernatant was taken and dye (10 µL, 2x) was added.
13. The protein samples were denatured using the heating block at 95° C.
14. The samples were cooled on ice (1 min) then loaded onto the gel (prepared according to SDS- PAGE standard protocol) and run at 150 V for 45 min.
