Difference between revisions of "Team:Manchester/Experiments"

This page shows general protocols given by the suppliers, supervisors or published papers. Visit Lab Book page for detailed experimental procedures (including trials and errors, attempted improvements etc) undergone by us.

Chemical Transformation

Materials (consumables):
LB broth (Luria Bertrani medium = rich media to grow bacteria)
TSS buffer (to prepare chemically competent cells)
S.O.C. medium (helps obtain the maximal transformation efficiency)
LB agar (gel where bacteria can grow)
Chloramphenicol (CAL) at stock concentration 25mg/ml

Preparation of chemical competent cells:

Inoculate DH5α cells into 50mL LB and incubate at 37°C
Monitor growth every 30 min by measuring optical density at 600nm (OD600) until it reaches OD600 = 0.4-0.6
Harvest cells and prepare using 'TSS competent E.coli protocol' shown below:

LB Agar plates preparation:

Prepare LB containing chloramphenicol (CAL) (at 25μg/ml)

Melt LB in microwave (defrost setting for 15 min)
Cool LB by running cold water over
Stock of 25mg/ml CAL → so add 400μl CAL to 400ml LB

Pour plates (in fume hood) and allow to solidify

Chemical Transformation:

Add 1μl of DNA to 50μl of competent cells, mix well and place on ice for at least 30 min
Heat shock cells at 42°C for 30 seconds, followed by 2 min incubation on ice
Add 450μl of SOC medium to the cells and incubate for 45 min at 37°C (to allow protein expression (particularly the antibiotic resistance gene)
Plate and spread (glass spreader sterilised over a flame and in ethanol) 50, 100, and 200μl of the cells into the agar plates made previously
Incubate at 37°C for 2 hours

TSS Competent E. coli Preparation

Materials (consumables):
Dimethyl sulfoxide (DMSO)
Polyethylene glycol (PEG)
MgCl2 stock solution
LB or SOC liquid medium
Ice

Methods:

Inoculate DH5α E. coli cells into 50ml LB broth and incubate at 37°C
Prepare the TSS buffer while waiting for the culture to grow and place it on ice (see: Table below)
Monitor growth of the culture every 30 min by measuring the optical density at 600nm wavelength (OD600) until it reaches OD600 = 0.4-0.6 (takes approximately 2-3 hours)
Once the proper optical density has been achieved, take 100ml of culture and centrifuge under 2.700xg for 10 min at 4°C
Resuspend each tube in 5ml of pre-chilled TSS buffer with gentle vortexing
Chill TSS suspended cells on ice for 15 min
Aliquot 200μL of TSS suspended cells while ensuring the cells remain well mixed
Cells can be used immediately or stored at -80°C

TSS Buffer Composition

Component	Stock (M)	Amount
MgCl2	2	0.300ml
DMSO	-	1ml
PEG (3350 or 8000)	-	2g
LB Medium	to final volume of 20ml	to final volume of 20ml

PCR From Plasmid DNA Template

Materials (consumables):
2x Q5 Master Mix
10 µM forward primer
10 µM reverse primer
DNA template
Nuclease-free water

Methods:

Gently mix the reaction from the components listed in the table below and place on ice
Note: addition of reagents are done in the following order to prevent degradation of primers: nuclease-free water, both primers, DNA, and then Q5 mix

PCR Reaction Components

Component	25µl Reaction	50µl Reaction	Final concentration
Q5 High-Fidelity 2X Master Mix	12.5µl	25µl	1X
10µM Forward Primer	1.25µl	2.5µl	0.5µM
10µM Reverse Primer	1.25µl	2.5µl	0.5µM
Template DNA	variable	variable	< 1,000ng
Nuclease-Free Water	to 25µl	to 50µl

When necessary, collect all liquid to the bottom of the PCR tube by spinning for a short time
Transfer the PCR tube from ice to a PCR machine and begin thermocycling

Thermocycling
The PCR machine should be set to run the following steps:

Step	Temperature (°C)	Time
Initial denaturation	98	30 seconds
30 cycles	98 (denaturation) 63 (annealing) see Note 1 72 (extension)	5 seconds 30 seconds 27 seconds per kb
Final extension	72	2 minutes
Hold	10	-

Note 1: The NEB Tm calculator should be used to determine the annealing temperature when using the Q5 Master Mix: http://tmcalculator.neb.com/#!/

Gel Electrophoresis

Materials (consumables):
1% TAE Buffer
Agarose powder
SYBR Safe
Loading dye
DNA ladder

Make 1% agarose gel:

Prepare 1% TAE agarose gel: dissolve 1g of agarose into 100ml of TAE buffer in a conical flask
Warm in microwave for 1 min at max power
Remove flask from microwave with care, swirl gently and cool under running tap
Add 5µl of SYBR Safe
Prepare a casting tray with suitable comb
Pour to cool mixture into the casting tray and wait 15 min until it solidifies

Run gel:

Add 5µl of PCR solution and 1µl 10x loading dye
Load 6µl of DNA ladder alongside and all samples (Do not forget to add dye to ladder too) - NEB 1kb ladder used
Run gel at 100V for 45 min
Visualise gel on a transilluminator (SYBR Safe binds DNA and fluoresces under UV light)

Restriction Digestion and Ligation

Restriction Digestion
Materials (consumables):
Restriction Enzyme: NEB enzyme finder to determine the restriction enzymes
10X Buffer: NEB double digest finder to determine the buffers that are required
Plasmid DNA
Nuclease-free water

Methods:

Incubate the digestion reaction (see components below) for 1 hour at 37°C, then at 65°C for 20 min (to heat-inactivate the enzymes)

Restriction Digestion Mix

Component	50µl Reaction	Final concentration
10X Buffer	50µl	1X
DNA	will vary	250ng
Restriction Enzymes	1µl (of each enzyme)	10U
Nuclease-free water	to 50µl

Ligation
Materials (consumables):
T4 Ligase Buffer
T4 Ligase
Vector DNA
Insert DNA
Nuclease-free water
Ice

Methods:

Allow the buffer to defrost on ice
Calculate molar ratio for vector and insert DNA using NEBioCalculator (NEBioCalculator) (ideal ratio for insert:vector is 3:1)
Make up ligation reaction as below

If using T4 ligase: incubate at room temp for 1 hour
NB: different ligases (eg. Quick Ligase) will require different incubation times

Ligation Reaction Composition

Component	10µl Reaction	Final concentration
10X T4 Ligase Buffer	1µl	1X
Vecor DNA	vary
Insert DNA	vary
Nuclease-free water	to 10µl

PCR Purification (QIAquick PCR Purification Kit)

Materials (consumables):
Ethanol (96-100%)
Buffer PE
Buffer PB
Buffer EB
pH indicator I
Sodium acetate
QIAquick column
Loading dye

Notes before starting:

Add ethanol (96-100%) to buffer PE before use (see bottle label for volume)
All centrifugation steps are carried out at 17,900 x g (13,000 rpm) in a conventional table-top microcentrifuge at room temperature
Add 1:250 volume pH indicator I to Buffer PB. The yellow color of Buffer PB with pH indicator I indicates a pH of ≤7.5. If the purified PCR product is to be used in sensitive microarray applications, it may be beneficial to use Buffer PB without the addition of pH indicator I. Do not add pH indicator I to buffer aliquots

Methods:

Add 5 volumes Buffer PB to 1 volume of the PCR reaction and mix. If the color of the mixture is orange or violet, add 10μl 3M sodium acetate, pH 5.0, and mix. The color of the mixture will turn yellow
Place a QIAquick column in a provided 2 ml collection tube
To bind DNA, apply the sample to the QIAquick column and centrifuge for 30–60 seconds until all the samples have passed through the column. Discard flow-through and place the QIAquick column back in the same tube
To wash, add 0.75ml Buffer PE to the QIAquick column and centrifuge for 30–60 seconds. Discard flow-through and place the QIAquick column back in the same tube
Centrifuge the QIAquick column once more in the provided 2ml collection tube for 1 min to remove residual wash buffer
Place each QIAquick column in a clean 1.5ml microcentrifuge tube
To elute DNA, add 50μl Buffer EB (10 mM Tris·Cl, pH 8.5) or water (pH 7.0–8.5) to the center of the QIAquick membrane and centrifuge the column for 1 min. For increased DNA concentration, add 30μl elution buffer to the center of the QIAquick membrane, let the column stand for 1 min, and then centrifuge
If the purified DNA is to be analyzed on a gel, add 1 volume of Loading dye to 5 volumes of purified DNA. Mix the solution by pipetting up and down before loading the gel

Miniprep (QIAprep Spin Miniprep Kit)

Materials (consumables):
Overnight culture
Buffer P1
Buffer P2
Buffer N3
Buffer PE
Sterile dH2O (MiliQ) water

Methods:

Spin down the overnight cultures at 10000rpm for 10 min. Discard supernatant into virkon
Resuspend the pelleted bacteria with 250μl of Buffer P1 (stored in fridge), and transfer the resuspended bacteria into a fresh 2ml eppendorf
Add 250μl of Buffer P2 to the 2mL Eppendorf with bacteria and mix gently. Sample should turn blue (indicates cells have lysed)
Incubate for 5 min at room temperature (do not exceed 5 min or plasmid will begin to degrade)
Add 350μl of Buffer N3 and mix gently. Sample should be colourless and contain a white precipitant
Centrifuge samples at 14,000rpm for 10 min using a table top centrifuge
Transfer 750μl of the supernatant to a column placed on a 1.5ml Eppendorf tube (discard white precipitate). Centrifuge at 11,000rpm for 1 min using a table top centrifuge
Discard the flow-through. Place column onto new eppendorf tube and add 750μl PE buffer (with added ethanol to the stock buffer if not already done so). Incubate at room temperature for 5 min. Centrifuge at 13,000rpm for 30 seconds
Transfer column to fresh eppendorf. Centrifuge at 13,000rpm for 2 min (dry out)
Transfer column into fresh eppendorf. Add 30μl of sterile dH20 (MiliQ) (add directly onto column to ensure water pushes DNA through) and incubate for 5 min at room temperature
Centrifuge at 11,000rpm for 1 min. Do not discard this flow-through. This contains the extracted plasmid

Gel Extraction (QIAgen Gel Extraction Kit)

Materials (consumables):
Buffer QG
Buffer PE
Buffer PB
3M Sodium Acetate
Sterile dH2O (MiliQ) water
Isopropanol

Methods:

Excise the DNA fragment from the agarose gel with a clean, sharp scalpel
Weigh the gel slice in a colorless tube. Add 3 volumes Buffer QG to 1 volume gel (100mg gel ~ 100μl). The maximum amount of gel per spin column is 400mg. For >2% agarose gels, add 6 volumes Buffer QG
Incubate at 50°C for 10 min (or until the gel slice has completely dissolved). Vortex the tube every 2–3 min to help dissolve gel. After the gel slice has dissolved completely, check that the color of the mixture is yellow (similar to Buffer QG without dissolved agarose). If the color of the mixture is orange or violet, add 10μl 3 M sodium acetate, pH 5.0, and mix. The mixture turns yellow
Add 1 gel volume isopropanol to the sample and mix
Transfer 750µl of supernatant to a column placed on a 1.5ml eppendorf. Centrifuge at 11,000 rpm for 1 min
Discard flow through. Place column in a new Eppendorf tube. Add 500µl of PB buffer and centrifuge column at 13,000 rpm for 30 seconds
Discard flow through. Place column in a new eppendorf. Add 750µl of PE buffer (check ethanol has been added, see: PCR Purification). Incubate at room temp for 5 mins, then centrifuge at 13,000 rpm for 30 seconds
Transfer column into fresh Eppendorf. Centrifuge at 13,000 rpm for 2 mins
Transfer column to new eppendorf. Add 30µl of MiliQ water and incubate for 5 mins at room temperature
Final centrifuge at 11,000 rpm for 1 minute
Test sample using Nanodrop (see below)

Nanodrop: calculate DNA concentration in sample

Load 1μL of MiliQ water to the Nanodrop and blank. Clean and load another 1μl of MiliQ water, measure and proceed only if clean (absorbance is zero at all measured wavelengths)
Load 1μl of sample and measure DNA concentration
Check A260/280 ~ 1.8 and A260/230 ~2.0 (pure DNA sample should have values close to these numbers)

LacUV5, Tet and araBad Promoter induction

Materials (consumables):
LB liquid medium
Chloramphenicol at stock concentration of 25mg/ml
IPTG at stock concentration of 100mM
Tetracycline at stock concentration of 0.214mM
Arabinose at stock concentration of 100nM

Method:
Aseptic technique maintained at all times

Transfer 50mL of LB +250L of chloramphenicol into 8x 250mL conical flasks.
Inoculate 2 flasks labelled + and - with 200L of pSBC13eutS overnight culture.
Grow the cultures at 37C in shaking incubator until and OD value of 0.5 is reached.
Add IPTG, Tetracycline and Arabinose to the respective constructs in the amounts shown below.

Inducers to be added to Eut constructs

Flask	Construct	Inducer and concentration to be added
Flask 1	pSBC13eutS-	None
Flask 2	pSBC13eutS+	250µM IPTG
Flask 3	pSBC13eutMN-	None
Flask 4	pSBC13eutMN+	1000 nM tetracycline
Flask 5	pSBC13eutSMN-	None
Flask 6	pSBC13eutSMN+	250µM IPTG and 100nM tetracycline
Flask 7	pSBC13eutLK-	None
Flask 8	pSBC13eutLK+	500µL arabinose

Incubate samples for 4hours in a 20C shaking incubator, remove 25mL of the culture and transfer to a falcon tube.
Centrifuge at 9000rpm for 8minutes.
Pour off supernatant, sample can be used immediately or frozen at -20C.
Incubate samples for a further 16 hours at 20C (20 hours in total), transfer culture to a falcon tube.
Centrifuge at 9000rpm for 8minutes.
Pour off supernatant, sample can be used immediately or frozen at -20C.

Expression analysis by Western blot, GFP and ?

Protocols.

Cell Fixation (using paraformaldehyde)

Materials (consumables):
Phosphate Buffered Saline (PBS)
PBS + 2% paraformaldehyde

Method:

Using a fresh transformation plate prepare overnights as follows:
10ml LB
10μl of each antibiotic as required (Chloramphenicol for localization tags and Ampicillin for Eut)
Inoculate media with a single colony
Grow overnight at 37⁰C
Prepare fresh liquid cultures as follows:
10ml LB
10μl of each antibiotic as required
Inoculate with 200μl of overnight culture
Grow to OD600 of 0.1, or for roughly 1 hour and induce the whole liquid culture with IPTG, Tet and/or Arabinose as required (IPTG for EutS, Tet for MN & Ara for LK. Tags are constitutive and don't need inducing).
Add 10μl of 100mM IPTG for 100μM, 2.34μl of 214μM Tet for 50nM & 10μl of 100mM Ara for 100μM.
Incubate induced samples at 30⁰C and take out at desired time-points (I found 2 and 3 hours are sufficient).
At each time-point remove 1ml of each liquid culture and place in 1.5ml eppendorfs.
Spin down at 5000rpm for 2 minutes in a table top centrifuge and discard supernatant.
Resuspend pellet in 1ml PBS and spin down again at 5000rpm for 2 minutes, discarding supernatant.
Next resuspend pellet in PBS+2% paraformaldehyde and leave at room temperature for 30 minutes.
After 30 minutes, spin down at 5000rpm for 2 minutes and discard supernatant and resuspend pellet in 100μl of PBS.

Preparing Microscope Slides

Materials (consumables):
Fixed cell suspension
Blank microscope slides
Cover slips

Method:

Pipette 12μl of suspension to a slide and add a cover slip.
Press cover slip down slightly (this helps put all cells in the same plane so it's easier to focus the microscope).
Allow to dry out of direct light to avoid photo bleaching (under blue roll is sufficient).
Store somewhere dark like a slide box or wrapped in lens cloth.

Image analysis (CellProfiler)

Analysis Modules:
ApplyThreshold
IdentifyPrimaryObjects
IdentifyPrimaryObjects
MeasureObjectSizeShape
ExportToSpreadsheet

Method:

http://cellprofiler.org/examples/

http://cellprofiler.org/tutorials/

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