Team:SCUT-China/Project/Protocol

CaCl2 competent cell

Materials

LB medium
Spectrophotometer
Shaking incubator at 37℃
Ice-cold CaCl2
15% glycerol
Icebox
-80℃ refrigerator

Protocol

• Inoculate 5ml LB with E.coli; incubate over night at 37℃ and 250 rpm.
• Inoculate 100ml LB with an aliquot of an overnight culture and grow to an O.D.600 of 0.3-0.4 at 37℃ with 250 rpm.
• Divide the cultures into two 50ml Epppendorf tubes.
• Keep on ice for 30min.
• Centrifuged at 4000rpm for 4min at 4℃.
• Discard supernatant.
• Resuspended in 10ml ice-cold CaCl2.
• Keeping on ice for 10min.
• Centrifuged at 4000rpm for 4min at 4 ℃ again.
• Discard supernatant.
• Resuspended in 10ml ice-cold CaCl2.
• Keeping on ice for 10min.
• Repeat the step 5-8 again.
• Centrifuged at 4000rpm for 4min at 4 ℃.
• Discard supernatant.
• Resuspended in 2ml CaCl2 containing 15% glycerol.
• Devide it into 1.5ml Eppendorf tubes, 100ul per tube.
• Stored at -80℃.

Heat-shock transformation

Materials

Plasmid or DNA ligation mix
Water bath 42℃
LB medium
Shaking incubator at 37℃
Eppendorf centrifugation
Selective plate

Protocol

• Add 1-2ul plasmid to 100ul competent cells. Homogenize by gently mixing with pipette several times. (Note that if the plasmid is product of linkage, add 10-20ul DNA)
• Keep on ice for 30min.
• Put it at 42℃ for 90s
• Keep on ice again for 2min.
• Add 400ul LB .
• Incubate the cells at 37℃ with 100rpm for 45min.
• Centriguged at 4000rpm for 1min.
• Remove 300ul of supernatant.
• Resuspended the rest of 200ul of cultures and plate it onto selective plates.

Eletrocompetent cell

Materials

LB medium
Spectrophotometer
Shaking incubator at 30℃
Ice-cold 10% glycerol
Icebox
-80℃ refrigerator

Protocol

• Inoculate 5ml LB with E.coli; incubate over night at 30℃ and 250 rpm. Our E.coli has transformed plasmid PKD46, which would lose at 37 ℃
• Inoculate 100ml LB with an aliquot of an overnight culture and grow to an O.D.600 of 0.6 at 30℃ with 250 rpm. When the O.D.600 is 0.2, add 3mmol arabinose to induce the PKD46 to produce the protein we need
• Divide the cultures into two 50ml Epppendorf tubes
• Keep on ice for 30min
• Centrifuged at 4000rpm for 4min at 4℃
• Discard supernatant
• Resuspended in 10ml ice-cold sterile water
• Keeping on ice for 10min
• Centrifuged at 4000rpm for 4min at 4 ℃ again
• Discard supernatant
• Resuspended in 10ml ice-cold 10% glycerol
• Keeping on ice for 10min
• Centrifuged at 4000rpm for 4min at 4 ℃ again
• Discard supernatant
• Resuspended in 10ml ice-cold 10% glycerol
• Keeping on ice for 10min
• Repeat the step 9-12 again
• Centrifuged at 4000rpm for 4min at 4 ℃
• Discard supernatant
• Resuspended in 1ml 10% glycerol
• Devide it into 1.5ml Eppendorf tubes, 100ul per tube
• Stored at -80℃

Electrotransformation

Materials

75% ethanol
Linear DNA fragement
SOC medium
Electroporation device
Icebox
Electrocompetent cell
Selective plate
Shaking incubator at 37 ℃
Eppendorf centrifugation

Protocol

• Taken from 75% ethanol, cuvette is exposed to ultraviolet ray for 30min in bechtop after drying
• Then keep the sterilized cuvette on ice
• Add 1-10ul(300ug) DNA to 100ul of electrocompetent cells
• Homogenize by gently mixing with pipette several times and then transfer mixture into a prechilled cuvette
• Wipe moisture from cuvette and insert the cuvette into the electroporation device
• Electroporation

Mode	Bacterial
Voltage	1.8kV
Time constant	6ms

• Immediately add 1ml of SOC medium
• Transfer the mix into a sterile 1.5ml tube
• Incubate at 37 ℃ with 150 rpm for 1h
• Centriguged at 4000rpm for 1min
• Discard 800ul of supernatant
• Resuspend the rest of 200ul of culture solution and plate it onto selective plates

SDS-PAGE

Materials

Stacking gel
Separating gel
Comb
SDS running buffer
Gel electrophoresis
Collodial coomassie

Protocol
Pouring the polyacrylamide gel

• Make a master mix for the stacking and separating gel without adding ammonium persulfate and TEMED
• Aliquote 6.5 mL for each separating and 2.5 mL for each stacking gel
• Add ammonium persulfate and TEMED to each separating gel aliquote
• Pour the solution quickly into the gel casting form. Leave about 2 centimeters below the bottom of the comb for the stacking gel
• Layer wateter on top of the gel
• Leave the separating gel at room temperature for 1h to polymerize
• Remove water and wait until the surface is dry
• Add ammonium persulfate and TEMED to each stacking gel aliquote and pour the solution quickly into your gel casting form
• Insert comb without getting bubbles stuck underneath
• Leave the gel at room temperature for 1h to polymerize
• Remove sealing and store the gel wrapped in moistened paper towel at 4°C

Running the gel

• Remove sealing, put the polymerized gel into gel box and pour SDS running buffer into the negative and positive electrode chamber
• Remove comp without destroying the gel pocket
• Pipet the sample into the gel pockets
• Connect the power lead and run the stacking gel with 10 mA until the blue dye front enters the separating gel
• Raise amperage up to 20 mA for running the separating gel
• When the distance of the lowest molecular weight standard lane to the gel end is down to 0.5 cm stop the electrophoresis by turning off the power supply

Collodial coomassie staining

• Agitate the staining solution at 37 °C over night to form the colloids
• After finishing the SDS-PAGE remove gel from gel casting form and transfer it in to a box
• Add 100 mL of the staining solution to your polyacrylamid gel
• Incubate the gel in the solution at room temperature until the protein bands got an intensive blue color. Shake the gel continuously during incubation
• Remove the staining solution
• Wash the gel with dH2O
• Incubate the gel in ddH2O (2-6 h) for bleaching the background
• Shake the gel continuously during incubation. If necessary replace the colored water with new one

Western blot

Materials

PBS
Eppendorf centrifugation
Protease inhibitor
Spectrophotometer
Sample buffer
Electrophorator
Sponge
PDVF
Methanol

Protocol
Protein extraction from adherent cells

• Wash cells in the tissue culture flask or dish by adding cold phosphate buffered saline (PBS) and rocking gently. Discard PBS
• Add PBS and use a cell scraper to dislodge the cells. Move the mixture into microcentrifuge tubes
• Centrifuge at 1500 RPM for 5 minutes
• Discard the supernatant
• Add 180 μl of ice cold cell lysis buffer with 20 μl fresh protease inhibitor cocktail. (Make sure the protein concentration is high enough, if not it is advised to repeat the procedure with a higher proportion of protease inhibitor cocktail)
• Incubate for 30 minutes on ice
• Clarify the lysate by spinning for 10 minutes at 12000 RPM at 4℃
• Transfer supernatant (or protein mix) to a fresh tube
• Measure the concentration of protein using a spectrophotometer
• store on ice or frozen at -20℃or -80 ℃

Sample preparation

• Put 50μg of protein extract in each well
• Add 5 μl sample buffer to the sample, and make the volume in each lane equalized using ddH2O. Mix the buffer and the sample
• Heat the samples with dry plate at 100℃ for 5 minutes

Electrophoresis

• Pour the running buffer into the electrophorator
• Place the gel inside the electrophorator and connect to a power supply
• Make sure the buffer covers the gel completely, and remove the comb
• Load 6μl of marker and 15 μl of samples into each well
• Run the gel with low voltage (60 V) for separating gel ; higher voltage (140 V) for stacking gel
• Run the gel around an hour, or until the dye front runs off the bottom of the gel

Electrotransfer

• Cut 6 filter sheets to fit the measurement of the gel, and one polyvinylidene fluoride (PDVF) membrane with the same dimensions
• Wet the sponge and filter paper in transfer buffer, and wet the PDVF membrane in methanol
• Separate glass plates and retrieve the gel
• Create a transfer sandwich as follows: Sponge, 3 Filter Papers, Gel PVDF, 3 Filter Papers.(Make sure there is no air bubbles between the gel and PVDF membrane)
• Relocate the sandwich to the transfer apparatus, which should be placed on ice to maintain 4℃
• Add transfer buffer to the apparatus, and ensure that the sandwich is covered with the buffer
• Place electrodes on top of the sandwich, ensuring that the PVDF membrane is between the gel and a positive electrode
• Transfer for 90 minutes

RNA extraction

Materials

water bath 65℃
AE
SDS
Phenol
Chloroform
Eppendorf centrifugation
RNase free water
75% ethanol
Absolute ethanol

Protocol

• Mix the 450ul AE,50ul SDS,350ul phenol and 350ul chloroform
• Pre-heated at 65 ℃
• Add 1ml the mixed liquor to resuspent the pelleted cells
• Shake violently
• Put it in 65 ℃ water bath evaportor
• Keep on ice for 5min
• Centrifuged with 1200g for 7min at 4 ℃
• The supernatant is transferred to a 1.5ml Eppendorf tube
• Mix with 600ul chloroform
• Centrifuged with 1200g for 7min at 4 ℃
• The supernatant is transferred to a new Eppendorf tube
• Add 1/10 volume NaAc and 2.5 times the volume absolute ethanol
• Keep at -20 for 30min
• Centrifuged with 1200g for 15min at 4 ℃
• Discard supernatant
• Resuspended with 1ml 75% ethanol
• Centrifuged with 1200g for 5min at 4 ℃
• Discard supernatant
• The tube is exposed at in bechtop for 2-3min to remove the liquid totally
• Add 20ul RNase free water to dissolve the RNA sediment

PCR

Real time PCR
Remove genome DNA

• Prepare 10ul reaction in 0.5ml PCR tube

Ingredient	Amount(ul)
5×gDNA Eraser Buffer	2
gDNA Eraser	1
Total RNA	*1
RNase Free dH2O	Up to 10

• Mix the reaction and spin down in microcentrifuge
• Put the tube in 42 ℃ water bath for 2min
• Stored in 4 ℃

Reverse transcription

• Prepare 20ul reaction in 0.5ml PCR tube

Ingredient	Amount(ul)
The above mixture	10
PrimeScript RT Enzyme Mix I	1
RT Primer Mix	1
5×PrimeScript Buffer 2(for Real Time)	4
RNase Free dH2O	4
Total	20

• Mix the reaction and spin down in microcentrifuge
• Put the tube in 37 ℃ water bath for 15min
• Then transfer the tube in 85 ℃ water bath for 5sec
• Stored in 4 ℃

Real time PCR

• Prepare 25ul reaction in 0.5ml PCR tube

Ingredient	Amount(ul)
SYBR Premix Ex TaqII(Tli RNaseH Plus)(2×)	12.5
Forward Primer(10uM)	1.0
Reverse Primer(10uM)	1.0
RT reaction	2
dH2O	8.5
Total	25

• Mix the reaction and spin down in microcentrifuge
• Run the PCR machine using bellowing conditions

Taq PCR

• Prepare 20ul reaction in 0.5ml PCR tube

Ingredient	Amount(ul)
λDNA(2.5ng/ul)	1
Forward Primer(10uM)	2
Reverse Primer(10uM)	2
2×PCR Mix	25
ddH2O	Up to 20

• Mix the reaction and spin down in microcentrifuge
• Run the PCR machine using bellowing conditions

KOD PCR

• Prepare 50ul reaction in 0.5ml PCR tube

Ingredient	Amount(ul)
dNTPs	10
2×PCR buffer for KOD FX	25
Forward Primer(10uM)	1.5
Reverse Primer(10uM)	1.5
KOD FX enzyme	1
Genome(plasmid orλDNA)	200ng(50ng or 50ng)
ddH2O	up to 50

• Mix the reaction and spin down in microcentrifuge
• Run the PCR machine using bellowing conditions

GC-MS

Materials

Ethanol
Chloroform
Water bath at 80 ℃
NaOH
Methanol
NaCl
GC-MS

Protocol
Sample Preparation

• Add 2ml ethanol and 1ml chloroform to a flask
• Add 2 drops of 6M HCl into the flask
• Refluxed at 80 ± 2℃ in water bath with stirring for 30 minutes
• After completing the reflux, extract with 1 ml of the mixture of diethyl ether and petroleum ether (1:1) for two times with a separatory funnel
• Collect the organic layer in the separatory funnel after every extraction
• Combined them into another round bottom flask, and evaporate in a water bath with stirring until near to dryness
• Add 2 ml of 0.5 M NaOH in methanol into the round bottom flask

Derivatisation

• Transfer 1ml of extract to another a screw cap test tube
• Add 1 ml of BF3
• The solution is refluxed for an additional 5 minutes
• After cooling down, add 1ml of the mixture of 0.5 μl/ml 1-bromotetradecane (the internal standard) in n-heptane and 1 ml of saturated sodium chloride solution
• Gently shake the screw cap test tube for several times and collect the upper layer into a vial for GC analysis

GC Analysis

• 1μl of sample solution is injected into GC. Make sure the GC is set using the following condition
• Set the automatic run for your samples using the autosampler
• Record the peak area and retention time of each analytical peak for calculation

Immunodetection

Materials
Alexa Floor 488
Fluorescence microscope
Shaking incubator at 37℃
1×PBS
Microscope slide
1% BSA

Protocol

• Take out 1ml E.coli which have been induced with IPTG
• Centrifuged with the speed of 6000g for 5 min
• Discard supernatant
• Add 1×PBS to resuspend the thallus
• Centrifuged with 6000g, 5 min
• Discard supernatant
• Add 1×PBS to resuspend the thallus
• Wash the thallus with 1×PBS like above for 3 times
• Add optimum amount TBS containing 1% BSA to resuspend the thallus and adjust the OD600 to 5
• Add 1.2ul monoclonal antibody to 200ul of cell suspension
• Incubate the mix at 37℃ for 2h
• Centrifuged the mix with 6000g for 5min
• Discard supernatant
• Add 1×PBS to resuspend the thallus
• Wash the thallus with 1×PBS like above for 3 times
• resuspended with 1×PBS containing 1% BSA
• Centrifuged the mix with 6000g for 5min
• Discard supernatant
• Resuspended with 1×PBS containing 1% BSA adding 1.2 ul Alexa Floor 488 which is an IgG antibody
• Incubate the mix at 37℃ for 1h
• Add 1×PBS to resuspend the thallus
• Discard supernatant
• Add 1×PBS to resuspend the thallus
• Wash the thallus with 1×PBS like above for 3 times
• Resuspended with 1.5ml 1×PBS
• Take out 2ul cell suspension to put on microscope slide and then observe it by fluorescence microscope

Digestion

Materials

plasmid DNA or PCR product
restriction enzymes
buffer (10x)
H2O
water bath at 37 °C
heat block or water bath at 65 °C

Protocol

• Prepare 20ul reaction in 0.5ml PCR tube

ddH2O	15ul
10×FastDigest Green buffer	2ul
Plasmid(PCR product or genome)	2ul(up to 1ug) or 10ul(~0.2ug) or 10ul(5ug)
FastDigest enzyme	1ul

• Mix the reaction and spin down in microcentrifuge
• Incubate for (at least) one hour at 37 °C
• Inactivate the restriction endonucleases by heat, incubation at 65 °C for 10 minutes and centrifuge shortly

Ligation

Materials

digested plasmid DNA or PCR product
T4 ligation buffer (10x)
T4 ligase
H2O
water bath at 16 °C

Protocol

• Prepare 10ul reaction in 0.5ml PCR tube

10×T4 buffer	2.5ul
T4 enzyme	1ul
DNA inserter	10 times to DNA vector
DNA vector	1/10 times to DNA inserter
ddH2O	Add to 25ul

• Mix the reaction and spin down in microcentrifuge
• Put it in16 °C water bath overnight

Gel running

Materials

Agarose
TAE 1x
SybrSafe DNA stain
Loading Dye
DNA ladder (Smartladder)
DNA electrophoresis machine

Protocol

• Dissolve agarose (w/v 0.6% for separating long DNA pieces (>10 kbp), 1% for separating shorter pieces) in 1x TAE by microwaving
• Close sides of electrophoresis tray (scotch tape works fine) and add comb
• Let solution cool and add 5 µl Sybrsafe to an empty electrophoresis tray (small gels) 10-12 µl Sybrsafe for larger gels
• Pour gel until a height of ~0.5 cm. Mix and remove bubbles with pipet tip (fast! It hardens quickly)
• Put tray into electrophoresis casing and add TAE until a small layer above the gel can be seen. Remove comb
• Add 1 µl loading dye to 5 µl sample, mix and load in the gel. Also add 5 µl smartladder for your reference
• Run gel on 80 V (long run)- 110 V (short run, mostly for a 'fast check') for ~40-60 minutes, dependant of gel size, separation acquisition and voltage

Material

LB medium

• 10 g Trypton
• 5 g yeast extract
• 10 g NaCl
• 12 g Agar-Agar (for plates)
• Adjust pH to 7.4

SOC medium
Add the following components for 900 ml of distilled H2O:

• 20 g Trypton
• 5 g Bacto Yeast Extract
• 2 mL of 5 M NaCl
• 2.5 ml of 1 M KCl
• 10 ml of 1 M MgCl2
• 10 ml of 1 M MgSO4
• 20 ml of 1 M glucose

Adjust to 1L with distilled H2O. Sterilize by autoclaving

TAE-Buffer
1 L of 50x TAE buffer

• 242.48 g Tris
• 41.02 g sodium acetate
• 18.612 g EDTA

Adjust pH to 7.8 with acetic acid
Solve in dH2O
Dilute 20 mL 50x stock in 1L dH2O for 1x Buffer for PAGE

SDS-PAGE gel
Stacking gel 5 %:

• 775 μL H2O
• 1.25 mL 0,25 M Tris (pH 6,8)
• 425 μL Bis/Acrylamide (0,8 %, 30 %)
• 50 μL 5 % SDS
• 25 μL 10 % Ammonium persulfate
• 3 μL TEMED

Separating gel 12 %:

• 1.5 mL H2O
• 2.8 mL 1 M Tris (pH 8,8)
• 3.0 mL Bis/ Acrylamide (0,8%, 30%)
• 150 μL 5% SDS
• 37.5 μL 10% Ammonium persulfate
• 5 μL TEMED