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AMPLIFICATION OF GENOMIC DNA

Cell Lysis

We used Stratagene kit for lysing human cells and extracting DNA.

• Pellet up to a maximum of 1x 106 cells for 30 sec. in microfuge. If more than 1X 106 cells are used, the reagent volumes will have to be increased accordingly. For adherent cells, a trypsin treatment is generally used prior to this step to free the cells into suspension. Be careful to perform the trypsin step in a timely manner so that5 the cells do not remain in undiluted trypsin for a long period of time.

• Aspirate media and wash cells with 500 l of 1x PBS.

• Pellet cells 30 sec. in microfuge at 14, 000 rpm.

• Aspirate PBS and resuspend cells in 500 l of PBS. Repeat spin and aspiration steps.

• Resuspend cells in 100 l PBS and 200l sterile water.

• Lyse cells by heating to 950C for 10-15 minutes.

• Allow the cells to cool briefly by setting at room temperature for 5 min. And add 10 l of 10 mg/ml Proteinase K to each sample. Quick vortex to mix.

• Incubate at 550C for one hour.

• Inactivate Proteinase K by heating to 950c for 10 minutes.

• Spin down condensation.

• Store at -200c.

PCR Amplification

Biolabs Phusion High- Fidelity DNA polymerase was used for all PCR amplifications. The reactions and conditions are given below.

Conditions:

PCR amplification with linkers required an additional 50 M Mg2+ in the mastermix.

USER Amplification

Procedure

The USER mix components are mixed (Table in materials).The PCR product must be purified before used in USER cloning

● 2 µl of the USER mix is transferred to PCR tubes.

● The PCR products is added in equal amounts of each and incubated for 40 minutes at 37°C and for 30 min at 25°C.

Site-Directed Mutagenesis

• Purify template plasmid DNA from a dam+ Escherichia coli strain (to ensure that all GATC sites are methylated for later digestion with DpnI).

• Design forward and reverse primers that will bind to the region of DNA you want to mutate but that contain the modifications you wish to make. See the CAD tool PrimerX.

• Run a primer-extension reaction with a proof-reading, non-displacing polymerase such as Pfu DNA polymerase. This results in nicked circular strands of the plasmid.

• Cut up the template DNA with DpnI.

• Transform the circular nicked DNA into a highly competent strain such as XL1-Blue. These cells will repair the nicks and not restrict the unmodified product DNA.

• Select colonies with the correct DNA.

Purification of PCR Products

QIAquick PCR Purification Kit Protocol using a micro centrifuge was followed for purification of PCR products.

Procedure

• Add 5 volumes of Buffer PB to 1 volume of the PCR sample and mix. It is not necessary to remove mineral oil or kerosene.

• Place a QIAquick spin column in a provided 2 ml collection tube.

• To bind DNA, apply the sample to the QIAquick column and centrifuge for 30–60 s.

• Discard flow-through. Place the QIAquick column back into the same tube. To wash, add 0.75 ml Buffer PE to the QIAquick column and centrifuge for 30–60 s.

• Discard flow-through and place the QIAquick column back in the same tube. Centrifuge the column for an additional 1 min.

• Residual ethanol from Buffer PE is completely removed by an additional centrifugation.

• Add 30 l of double distilled nuclease free water to elute the DNA and centrifuge again.

• Collect the flow through.

ANALYSIS OF DNA

Gel Electrophoresis

• Depending on the expected size of the fragments to be analysed, the percentage of gel is determined. For fragments more than 1.2 kb, a 1% gel and fragments less than 1.2kb 2% gel is used.

• To make a 1% gel, 0.7g of agarose is added to 70 ml of TAE buffer.

• The agarose is melted in a microwave until the solution becomes clear.

• The solution is cooled down until it can be held for 5 continuous seconds.

• 1 µl of ethidium bromide is added to the solution.

Electrophoresis Setting

• Ensure electrophoresis chamber is clean and dry, tape the sides (with Autoclave tape, NOT standard masking tape) to make watertight. Slot in the desired comb.

• Add gel solution to the chamber, and wait to set. The comb can then be removed from the chamber.

• Fill the electrophoresis apparatus half-full with 1x TAE buffer solution avoiding air bubbles.

• The gel is loaded with the samples, a negative sample, and the DNA ladder.

• Connect the electrodes to the apparatus. Set DC voltage at 100V and run for around 60 minutes (or until DNA separates sufficiently)

• View the gel in a gel exposer.

Gel Extraction

We used the QIAgen quick gel extraction kit; the protocol is adapted from the QIAquick Gel Extraction Kit Protocol.

Protocol

• The gel is exposed to blue-light to illuminate the DNA fragments (stained by ethidium bromide).

• The desired DNA band is identified and physically removed with a knife, cleaned with ethanol.( ensure to wipe the ethanol off the knife to avoid degradation of DNA)

• Add 3 volumes of buffer QG to 1 volume of gel. E.g.add 300 μl of Buffer QG to each slice of gel (volume of the slice is 100 μl, and weighs approximately 100 mg).

• Incubate at 52°C for 10 min (or until fully dissolved). To aid solvation, mix by vortexing the tube every 2–3 min during the incubation. When the gel is fully dissolved, ensure that the color of the mixture is yellow, same as that of the QG buffer.

• Add 1 gel volume of isopropanol to the sample and mix.

• Place a QIAquick spin column in a provided 2 ml collection tube.

• Apply each sample of DNA fragment to a QIAquick column, and centrifuge for 1 min at 13000 rpm.

• Discard flow-through and place QIAquick column back in the same collection tube.

• To wash, add 0.75 ml of Buffer PE to QIAquick column and centrifuge for 1 min.

• Spin the column again to get rid of any residual ethanol.

• Add 30 ml of nuclease free water at the centre of the column and keep for 1 minute.

• Place the column in a collection tube and spin for 1 minute at 13000 rpm. Collect the flow through.

TRANSFORMATION OF BACTERIAL CELLS

Preparation of Antiobiotic Resistnt Plates

• 6.2 g of LB agar is dissolved in distilled water and dissolved.

• This is autoclaved and sealed for future uses.

• Agar is melted in microwave for 5 minutes, or until dissolves.

• Respective volume of required antibiotic is added to the solution after it has started cooling down.

• 20-20 ml of this is evenly poured into plates and allowed to cool.

Transformation

• DHα5 competent cells are taken out from -800c and thawed in ice for 5 minutes.

• 1 ml of SOC is incubated at 370C.

• 10 µl of required plasmid/part is added to the competent cells and subjected to heat shock at 420c, in a water bath for 1 minute to enable transformation.

• Placed back in ice for 5 minutes.

• 250 µl of preheated SOC is added to this and placed in a shaker at 370C, 200 rpm for 16 hours.

• The broth from the shaker is poured on to prepared plates with corresponding antibiotic resistance, under sterile conditions.

• 8 glass beads are added to the plates and slowly shaken to ensure even spreading.

• The glass beads are recycled.

• The plates are incubated overnight at 370C.

E.coli Cell Culture

• This is the method for overnight preparation of a 10ml cell culture of antibiotic-resistant E. coli. The plasmid incorporated in the bacteria ensures that it is resistant to a corresponding antibiotic, thus the growth media will have to be provided with a suitable volume of the antibiotic.

• The plasmids we used in our lab were mostly resistant to chloramphenicol, Ampicillin, and Kanamycin.

• The media was inoculated( with a sterile loop) with a single colony of the tarnsformant, under sterile conditions and placed in an incubator for 16 hours, at 370c.

• Before further extraction, a glycerol stock of the culture was made using 1ml of media and an appropriate volume of glycerol and stored at -800c for viability.

Extraction of DNA

We used QIAprep Spin Mini Prep Kit, the protocol is adapted from the QIAprep Mini Prep Handbook QIAprep Mini Prep Handbook.

• Resuspend pelleted bacterial cells in 250 μl Buffer P1 and transfer to a microcentrifuge tube. Ensure that RNase A has been added to Buffer P1. No cell clumps should be visible after resuspension of the pellet. If LyseBlue reagent has been added to Buffer P1, vigorously shake the buffer bottle to ensure LyseBlue particles are completely dissolved. The bacteria should be resuspended completely by vortexing or pipetting up and down until no cell clumps remain.

• Add 250 μl Buffer P2 and mix thoroughly by inverting the tube 4–6 times. Mix gently by inverting the tube. Do not vortex, as this will result in shearing of genomic DNA. If necessary, continue inverting the tube until the solution becomes viscous and slightly clear. Do not allow the lysis reaction to proceed for more than 5 min.

• Add 350 μl Buffer N3 and mix immediately and thoroughly by inverting the tube 4–6 times. To avoid localized precipitation, mix the solution thoroughly, immediately after addition of Buffer N3. Large culture volumes (e.g. ≥5 ml) may require inverting up to 10 times. The solution should become cloudy.

• Centrifuge for 10 min at 13,000 rpm (~17,900 x g) in a table-top microcentrifuge. A compact white pellet will form.

• Apply the supernatants from step 4 to the QIAprep spin column by decanting or pipetting.

• Centrifuge for 30–60 s. Discard the flow-through.

• Recommended: Wash the QIAprep spin column by adding 0.5 ml Buffer PB and centrifuging for 30–60 s. Discard the flow-through. This step is necessary to remove trace nuclease activity.

• Wash QIAprep spin column by adding 0.75 ml Buffer PE and centrifuging for 30–60s.

• Discard the flow-through, and centrifuge for an additional 1 min to remove residual wash buffer. Important: Residual wash buffer will not be completely removed unless the flow-through is discarded before this additional centrifugation. Residual ethanol from Buffer PE may inhibit subsequent enzymatic reactions.

• Place the QIAprep column in a clean 1.5 ml microcentrifuge tube. To elute DNA, add 30 μl double distilled nuclease free water to the centre of each QIAprep spin column, let stand for 1 min, and centrifuge for 1 min.

MAMMALIAN CELL CULTURE

Thawing

• MG63 cells were derived from banked stocks at the ATCC (American type culture collection). The epithelial breast cancer cell line MCF7 used was a gift from Dr Miriam Dwek (University of Westminster)

• Vials were quickly defrosted in a 37 ˚C water bath.

• Re-suspended in 5 ml of complete media.

• Centrifuged at 1000 rpm for 3 mins.

• Supernatant was poured off and cells were re-suspended in 10 ml complete media and seeded in a 75 cm2 tissue culture flask.

Passaging Cells

Upon reaching the required confluency (70-90 %) cells were passaged as follows:

● Media discarded using a sterile pipette

● Washed once with Dulbecco’s phosphate buffered saline (DPBS) free from calcium and magnesium

● To detach cells from flask 2 ml of EDTA-trypsin was added and incubated at 37 ˚C for 2 minutes

● Upon cell detachment EDTA-trypsin was neutralised with 4 ml of complete media

● Centrifuged at 1000 rpm for 3 mins.

● Supernatant was discarded and cells re-suspended in complete media and seeded in to a fresh flask. Depending upon the cell line cells were passaged either at 1:3 or 1:6.

Freezing

● Cells trypsinised (as in passaging procedure above).

● Centrifuged at 1000 rpm for 3 mins.

● Supernatant poured off and cells re-suspended in FBS + 10 % DMSO and kept at -80 °C for 24 hours then transferred to liquid nitrogen.

Transferring Cells to Coverslips

● The filter of a 1 ml pipette is broken off, and the pipette is attached to the vacuum suction machine. The medium is removed, and the pipette is put in a 50 ml vial for later use.

● 1,5 ml 0,05% EDTA-trypsin/PBS is added to wash the cells. Tilt the flask quickly, so the EDTA-trypsin covers the cells. Remove the liquid quickly.

● 1,5 ml 0.05% trypsin-EDTA/PBS is added and the flask is incubated for 3 min at 37°C and 5 % CO2.

● 9 ml complete medium is added to inactivate the EDTA-trypsin and to wash the cells of the surface. Make sure the cells are well re-suspended, before you transfer them.

● Calculate the concentration of cells you want in the wells.

● Cover slips are placed in the bottom of a 6-well plate (2 cover slips/well). 2 ml cell suspension is added gently to each well.

● The plate is placed in the incubator at 37°C and 5% CO2 O/N.

Transfection of Cells (PEI Procedure)

All cell culture and transfection was carried in complete media Dulbecco’s modified eagles media (DMEM) supplemented with 10 % foetal bovine serum (FBS). No antibiotics were used

• One day prior to transfection 250000 MCF7 cells were seeded per well of a 24 well plate.

• For each transfection reagents were prepared as follows:

a) Plasmid mixed with PEI.

b) 100 µl (10% of growth media volume) 0.15M NaCl was added to plasmid-PEI mixture

c) Transfection reagent mixture was vortexed and incubated at room temperature for 10 mins.

• Prior to transfection media from each well was replaced and PEI-plasmid-NaCl mixture was added dropwise to cells.

• Cells were incubated overnight at 37ᵒC.

• Media replaced

• Cells allowed to recover for 48hrs then trypsinised and anlaysed as follows:

a) Media removed

b) Cells washed once with PBS

c) Trypsinised with 0.21 mM trypsin containing 4.81 mM EDTA

d) Trypsin was neutralized with complete media and cells centrifuged at 2000 rpm for 3 mins

e) Cells washed once in ice cold PBS containing 1% foetal bovine serum (FBS).

f) Cells re-suspended in 1% FBS containing 1ug/ml propidium iodide.

g) Cells analysed using CyAn™ ADP flow cytometer (DakoCytomation

In order to distinguish between alive and dead cells propidum iodide was used and data was analysed using the summit v4.3 software. Cell lines were gated according to an unstained sample and lasers were adjusted accordingly. Unstained cells were used to set gates for cell size and internal complexity, dead cells were removed along with doublets.

Aldehyde Dehydrogenase (ALDH) Staining

● Upon reaching a suitable confluency (70 - 90 %) cells were trypsinised and then neutralised using complete media (as in passaging procedure).

● Counted using a haemocytometer.

● Centrifuged at 1000 rpm for 3 mins.

● Resuspended in 0.5 ml of ALDEFLUOR buffer.

● An appropriate volume of cells is transferred to a separate eppendorf so each sample contains 5 x 105 cells (diluted in ALDEFLUOR buffer).

● 2.5 μl of the ALDH activated reagent was mixed with sample.

● 250 μl of sample was immediately removed and mixed in a separate eppendorf with 2.5 μl of the ALDH inhibitor diethylaminobenzaldehyde (DEAB) to act as a negative control.

● Samples were then incubated at 37 °C for 50 mins.

● Centrifuged at 2000 rpm for 5 mins and re-suspended in 0.5 ml ALDH buffer and analysed using a flow cytometer.

MICROSCOPY

● A 10X PBS dilution is made from PBS and MilliQ water.

● The coverslips are transferred to a 24-well plate (2 coverslip/well).

● The coverslips are washed by adding 1 ml diluted PBS to the wells. The PBS is discarded. This is done twice.

● 400 μl Formaldehyde is added to each well under the fume hood, and incubated for 12 min at RT. The liquid is transferred to the waste bin.

● The coverslips are washed 3 times with the diluted PBS.

● The coverslips are dipped in MilliQ water before laid on lint-free paper for drying.

● 4 drops each of 4 μl Vectashield is placed on a glass slide.

● When a coverslip is completely dry, it is placed on a drop of Vectashield on the glass slide. The procedure is repeated for each coverslip.

● The coverslips are fixated with transparent nail polish.