1) To a mixture of NaCl (5g), tryptone (5g), agar (7.5g) and yeast extract (2.5g), 250ml of distilled water was added and mixed in well.
2) An additional 250ml of distilled water was added.
3) This was heated till boiling.
4) These were autoclaved at 121ᵒC, 15psi for 10mins.
5) Into warm media (~50°C) antibiotic at the required concentration was added.
6) The media was then poured into labelled plates.
7) Plates were stored in the fridge until needed when hardened.
Flow Cytometry
1) Grew transformed Ecoli competent cells in an overnight culture containing 0, 1 or 10mM of Potassium Nitrate.
2) Pellet the cells then fix them in 4% PFA (500µL)
3) Spin down and resuspend in PBS (500µL)
4) These were then put into the Flow Cytometer for analysis:
BM-RFP transformed Ecoli were analysed using a BD Acuri C6 flow cytometer. Laser 488nm / Filter FL2-585
MB-CFP samples were analysed using a BD FACSAria II Cell Sorter. Laser 405nm. Detect V/450/40-A
Fluorometry Testing
1) Samples planned for testing we grown overnight at 37 degrees Celsius.
2) Samples of 5ml were pelletted into an eppendorf tube.
3) The pellets were resuspended using Tris buffer (1mL).
4) The cells were lysed using sonification, The sonifier was cleaned using ethanol. The samples were put in ice for 1 minute between each sonification. Each sample was sonified 5 times. Each time for 10 secs. Note: Be careful that the eppendorf does not touch the sonifier as it may shatter the eppendorf.
5) These were then centrifuged at a cold temperature for 10mins.
6) Following centrifugation the clear liquid was pipette off into fresh eppendorfs.
7) These samples were then diluted with Tris buffer (1500µL of Tris to 500µL sample).
8) Depending upon the reporter protein attached, the emission and excitation spectrums were different. For CFP, an excitation of 410nm and an emission wavelength scan of of 440nm to 500nm. For RFP an excitation of 560nm and an emission spectrum of 600-650nm was measured.
9) The fluorometer was set up using Tris buffer.
Gel Electrophoresis
1) An agarose gel (1% w/v) is made through 1g of agarosse mixed in with 100ml of 1X TAE buffer which is heated to dissolve before the addition of 5µL of ethidium bromide when hand holdable warmth. (Ensure this is in a fume cupboard with the relevant safety equipment). Depending on the fragment sizes expected different densities of gel were made. The smaller the fragments, the denser the gels.
2) Tray is assembled through taping the ends. After addition of agarose put in the comb to create wells. The agarose mixture was poured into this.
3) When dry, this was put into the electrophoresis machine and the wells filled.
4) Electrophoresis was run for 1 hour to 1 and a half hours at 120V.
Hydrating Dry DNA (Synthesised DNA)
1) The dry form synthesised DNA was centrifuged for 1min at 14500 rpm.
2) To these tubes, 20 µL of sterilised water was added.
3) These tubes were vortexed briefly (30secs) and centrifuged briefly (20secs).
4) These were then placed in ice.
Inoculations
1. To 5ml samples of sterilised LB broth antibiotics were added. Ampicillin was added at 500 μl and chlorophenicol was added at 3.75ml.
2. To each of these broths, E.coli colonies growing on plates was inoculated.
Isolating DNA (Bioline)
(Using Bioline ISOLATE Plasmid DNA Mini Kit and following the given instructions for low copy plasmids)
1) The culture was centrifuged at 14500rpm for 1 min to obtain a cell pellet.
2) The supernatant was discarded and the pellet resuspended with 500 µL of Resuspension Buffer using a vortex mixer or pipetting.
3) To the sample, 500 µL of Lysis Buffer P was added and mixed by inversion 5 times.
4) An aquilot of 600 µL of Neutralisation Buffer was added and the sample inverted 5 times.
5) To obtain a cell debris pellet the solution was spun at 14500rpm for 10 mins and the supernatant transferred to a collection tube.
6) After 1 min of centrifugation at 12000rpm the filtrate was discarded and 500 µL of Wash Buffer AP was added.
7) Another round of centrifugation at 12000rpm for 1 min was followed by the discard of the filtrate and the addition of 700 µL Wash Buffer solution.
8) The sample was centrifuged at 12000rpm for 2 mins and the spin column placed in an elution tube.
9) To the tube 100 µL of Elution Buffer was added and the sample incubated at room temperature for 1 min.
10) The isolated DNA was stored in eppendorf tubes and refrigerated at 4 ºC for later use.
LB Media
1) To a mixture of NaCl (5g), tryptone (5g) and yeast extract (2.5g), 250ml of distilled water was added and mixed in well.
2) An additional 250ml of distilled water was added.
3) This was heated till boiling.
4) The media was then transferred into culture flasks (5.5ml each) with the lids partially closed.
5) These were autoclaved at 121ᵒC, 15psi for 10mins.
Ligation
Digest vector and insert with relevant enzymes.
Use the equation to calculate ng of insert required for different ratios:
3 insert: 1 vector
1 insert: 1 vector
1 insert: 3 vector
Ng vector x bp size of insert x ratio of insert (e.g 3) = ng insert
Bp size of vector ratio of vector (e.g 1)
For a 20μl total reaction:
2μl T4 DNA ligase
2μl T4 DNA ligase buffer
16μl insert : vector ratio
Leave at room temperature overnight
Potassium nitrate solution (1M Stock Solution)
1) 20.2g of potassium nitrate was dissolved in distilled water.
2) The dissolved solution was made up to 200ml.
3) This was autoclaved at 121ᵒC, 15psi for 10mins.
Purification by Centrifugation
Using Promega Wizard SV Gel and PCR Clean-Up System kit
1) Using 10µL of membrane binding solution per 10mg of gel slice ratio, the gel slice was dissolved.
2) The dissolved solution was added to an SV minicolumn inside a collection tube.
3) This was left for 1min at room temperature.
4) This was centrifuged for 1 min at 14 000 rpm.
5) The overflow was discarded.
6) Membrane Wash Solution (700µL)containing ethanol was washed through and centrifuged for 1 min at 14 000 rpm.
7) The overflow was discarded and Membrane Wash Solution (500µL)was washed through and centrifuged for 5mins at 14 000 rpm.
8) The overflow was discarded and recentrifuged with the lid off for 1min at 14 000 rpm.
9) Nuclease Free Water (50µL) was added to the SV column which had been transferred from a collection tube to an eppendorf tub. This was left for 1 min at room temperature before centrifugation for 1min at 14000rpm.
10) This was stored in a fridge.
Restriction Digest
For multiple samples which need to be digested with the same enzymes, a master mix was created, which involved assembly of buffer, BSA and enzymes at a higher amount than that needed. This saved time decreased the inaccuracy of pipetting very small quantities.
Note: The buffer is chosen through maximising the efficiency of the enzyme or enzymes.
Single Digest for validation
1) A mastermix of a multiplication of the following amounts were set up in an eppendorf and mixed through pipetting up and down then briefly centrifugation: buffer (2µL), acetylated BSA at concentration of 10µg/µL (0.2µL) and restriction enzyme (0.5µL) were assembled in an ependorf. Direct assembly was made if only one sample was needed.
2) Into another eppendorf, DNA (1µL) and sterilised and deionised water (16.3µL) was added.
3) From the master mix, 2.7µL was pipetted into the DNA and water mixture.
4) The mixture was mixed through pipetting up and down with restriction enzymes.
5) The tube was centrifuged briefly.
6) This was incubated at 37ᵒC for 2-3 hrs.
Double Digest for isolation of a fragment
1) A mastermix of a multiplication of the following amounts were set up in an eppendorf and mixed through pipetting up and down then briefly centrifugation: buffer (2µL), acetylated BSA at concentration of 10µg/µL (0.2µL) and restriction enzymes (0.5µL of each) were assembled in an ependorf. Direct assembly was made if only one sample was needed.
2) From the master mix, 3.2µL was pipetted into separate eppendorfs.
3) Into these tubes, 1µg of DNA and water that makes each sample up to 20µL is added. The water is added first.
4) The mixture was mixed through pipetting up and down with restriction enzymes.
5) The tube was centrifuged briefly.
6) This was incubated at 37ᵒC for 5 hours to overnight.
Transfection
Mammalian Transfection MB-CFP and NO cytotoxicity Assay
Reagents:
Media:
. DMEM (Invitrogen #21885) without serum (SFM-DMEM) or with serum DMEM #21885 + 10% Fetal Calf Serum.
. DNA is Mammalian-Bacterial – Cyan Fluorescent Protein, Hybrid NO sensor MB-CFP.
. LipoD293 Transfection reagent: (SignaGen #SL100668)
. Nitric Oxide donor: SNAP S-Nitroso-N-Acetylpenicillamine (SNAP), 25 mg (Molecular Probes® #N-7892)
. SNAP (Stock 25mg/ml DMSO) 500µM final conc.
17/9/12 Transfect MCF7 cells with MB2-CFP
Transfect MCF7 cells with MB2-CFP
Transfection to test MB-CFP in mammalian systems
. MCF7 (Human Breast cancer cell line) seeded into 6 channel slide (Ibidi µ-Slide VI0.4 #80606) at 30µl of 3x105 cells/ml dilution on 16/9/12
.On day of transfection (17/9/12) make Tube 1 (50ul SFM + 3µl LipoD) Tube 2 (50ul SFM + 0.5µg DNA)
. DNA for transfections
o MB2-C11a/b (MB-CFP miniprep 1) 500ng = 6.5µl
o MB2-C12a (MB-CFP miniprep 2) 500ng = 6.5µl
. Scrape tubes / vortex to mix well
. Add Tube 1 (LipoD) to Tube 2 (DNA)
. Scrape tubes / vortex to mix well and leave for ~15min.
. Meanwhile remove media from cells, wash 100µl SFM DMEM
. Add 50µl Transfection mixture to relevant channel of Ibidi 6-channel slide.
(Each channel is transfected with 250ng of DNA.)
. Remove media 6 hours post transfection and replace with 150µl DMEM+FCS.
.9am next day (18/9/12) added SNAP 500µM final concentration.
. ~24h post transfection cells imaged on CCD2 inverted
NO Cytotoxicity Assay
. Assay to test cytotoxicity of SNAP Nitric Oxide donor on MCF7 cells.
. MCF7 cells (Human Breast cancer cell line) seeded into 6 well plate (250,000cells/well) on 16/9/12
. SNAP added at 500µM final conc. in DMEM+FCS on 18/9/12.
. Cells counted 24h post addition of SNAP (19/9/12) i.e. 72h post seeding.
General Protocol: Counting Cells
. Suspend cells in media after trypsin. Extract 25ul and dilute (1:1) in 25ul of trypan blue (detects dead cells). Prepare Hemacytometer and carefully squirt samples into chambers.
. Place under microscope and count cells in central chamber (5x5 grid). Cells on
border are counted too. Dead blue cells are not counted.
X (cell count) x 2 (dilution factor) x 104 = cells / ml
e.g. 42 x 2 x 104 = 840,000 cells per ml
. To calculate volume with desired number of cells.
No cells needed/cells per ml e.g. 200,000 / 840,000 x 1000 (ul) = 238ul/well
Transformation
1) Competent cells were thawed on ice for 15mins
2) Into pre-chilled 2ml tubes, 50 µL competent thawed cells and 2µL of re-suspended DNA were added and mixed through flicking.
3) These were put back into ice and left for 30mins.
4) From the ice, the tubes were taken and immersed in a 42 ºC water bath for 1 min.
5) After heat shocking, cells were incubated on ice for 5mins.
6) To these cells, 200 µL of pre-warmed LB broth (37 ºC) was added.
7) Cells were then incubated at 37 ºC for 2 hours and shaken every 5mins
8) From each tube two inoculations were made. 1) 20 µL of cells plus 180 µL of LB on chloramphenical LB agar plates, 2) 200 µL of cells. Agar plates contain chloramphenicol at a concentration of 50µg/ml.
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