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Protocols
SOPS (Standart Operating Procedures)
Phageproduction
Inoculate from glycerolstock or preculture 60ml DYT with Cm (25µg/ml) and Tet (20µg/ml) up to OD600 0.07
Let grow at 37°C up to OD600 0.3(-0.4)
Infection with VCS M13 (ca. 10 phages per cell)
Incubate for 10 min without shaking
Add 1mM IPTG Transfer culture to 28°C- shaker
After 1 h add Kan (70µg/ml)
After 4-5h centrifuge 50ml of cultures down (5000g, 15min, 4°C)
Decant supernatant into new 50ml-Falkon and centrifuge (5000g,15 min, 4°C)
Leave upper 40ml of supernatant + 8ml PEG/NaCl over night on ice in 4°C room
Centrifuge down phage precipitation(5000g, 45min, 4°C)
Discard supernatant, centrifuge down shortly(~30 sec)
Carefully pipette off the rest of supernatant (filtered pipette tips!)
Resuspend pellet in 1ml TBS and transfer into new 1.5ml Eppi
Centrifuge sample down at 4°C, 12min with max. speed
Transfer upper 95% into a new Eppi, add 200µl PEG/NaCl
Incubation on ice for min. 1h
Phage precipitation, centrifuge at 4°C 10 min with max. speed
Pipette off the supernatant
Resuspend pellet in 100µl TBS
Centrifuge sample at 4°C, 12 min with max. speed
Transfer upper 90µl in new Eppi
ELISA
Coating: add 100µl/well Streptavidin (1:1000) in NaCo3 and 100µl/well 1%BSA in TBS to a 96 well plate; mark the positions on a separate paper in a table
Incubate over night at 4°C gently shaking
Blocking: pour off coating solutions, add 350µl/well 1%BSA in TBS-CaCl2; incubate at 20°c by gently shaking for 2h
5x washing: add 200µl/well TBS-T (0.05%; 1mM CaCl2) and pour solution off 5x
Incubation: add 75µl/well phages (1x10^10 phages/well) in TBS-CaCl2;-buffer (TBS-CaCl2)"blank"; Incubate for 1h at 20°C(gently shaking)
5x washing: add 200µl/well TBS-T (0.05%; 1mM CaCl2) and pour solution off 5x
Antibody: add 75µl/well of 1:1000 dilution of anti- M13 mAb/HRP (in TBS-CaCl2); incubate for 1h gently shaking
5x washing: add 200µl/well TBS-T (0.05%; 1mM CaCl2) and pour solution off 5x
Substrate: add 75µl/well 1mg/ml ABTS; measure Absorbance immediately and after 3 and 5 min; 405nm
Chemical Transformation
Thaw competent cells at room temperature (note: biological material does not like ice water transition; if cells are not in an 2 ml Eppi put them in a 2 ml Eppi): (100 µl); add DNA (note DNA can also be added to frozen cells): ligation mixture approx. 5 µl or plasmid approx. 1 µl; DNA and cells: mix gently by shaking or slow pipetting; Incubation on ice for: 20-30 min; Heat shock at: 42°C for approx. 40 s; Optional: cool on ice for: 5 min; add sterile LB(or 2YT)medium (note: medium can be supplemented with extra salts) 900 µl; Incubation in orbital shaker (note: Eppis are easiest handeled when put in a glass test tube and secured with tape) at 37°C for approx. 60 min (note: ampicillin resistance emerges after about 30 min, chlorampehnicol resistance after >= 60 min); Plate cells on agar plates plates supplemented with the antibiotic selecting for the resistance marker of the transformed plasmid : e.g. ampicillin; plasmid: 10µl cells + 40µl LB; ligation: 2 plates: 1. 50µl cells 2. centrifuge @ 2000 rpm 3 min, discard supernatant, resuspend the cell pellet and plate *********** Text for lab journal: Transformation **********************
Transformation
experiment date: §§§ time: §§§
name of investigator: §§§
plasmid: name: §§§ number: §§§; production date: §§§; origin: §§§
ligation: new plasmid name: §§§; ligation date: §§§; origin: §§§; (optional vector name: §§§ insert name: §§§)
competent cells: strain name: §§§ date: §§§; origin: §§§; (optional competency: §§§ clones/µg)
incubation on ice for: §§§ min
(optional) cold shock: §§§ min
heat shock: §§§ s
addition of medium type: §§§; volume of medium: §§§ µl
incubation in shaker: §§§ min
plates: medium type: §§§ (LB, 2YT, SOB); antibiotics: §§§ (Amp, Cm, Tet, Kan); glucose: §§§%; date poured: §§§; origin: §§§
volumes plated on plate 1: §§§, plate 2: §§§; (optional) plate 3: §§§
plates labeled with strain, plasmid, name, date, plated volume: yes no
time finished plating: §§§
incubation of plates: place §§§, temperature: §§§
additional comments or observations:
Evaluation of Transformation (note: put in lab journal at the date of evaluation)
experiment date: §§§ time: §§§
name of investigator: §§§
cells: §§§ ; plasmid:§§§
transformation date: §§§, page in lab journal transformation experiment: §§§
visual inspection
colony size: §§§ (tiny <0.2 mm, small 0.2-1mm, normal 1-2mm, big >2mm)
colony size distribution: §§§ (homogeneous, varied, significantly varied)
shape of colonies: §§§ (round, rough edged)
number of colonies plate 1: §§§ ; fraction of plate counted: §§§
number of colonies plate 2: §§§ ; fraction of plate counted: §§§
additional observations:
conclusion:
************ End Text for lab journal: Transformation *****************
Plasmid Purification
Use QIAprep Spin Miniprep Kit; Centrifuge cells: @ 8000 rpm, 3 min; Discard supernatant; Resuspend pellet in Buffer P1: 250µl; Transfer to a microcentrifuge tube; Add Buffer P2: 250 µl; Mix by inverting the tube 4-6 times; Add Buffer N3: 350 µl; Mix by inverting the tube 4-6 times; Centrifuge: 10 min @ 13000 rpm; Apply supernatant to QIAprep spin column; Centrifuge: @ 13000rpm, 60 s; Discard flow-through; Wash QIAprep spin column by adding: 0.5 ml Buffer PB; Centrifuge: @ 13000rpm, 60 s; Discard flow-through; Wash QIAprep spin column by adding: 0.75 ml Buffer PE; Centrifuge: @ 13000rpm, 60 s; Discard flow-through; Centrifuge: @ 13000rpm, 60 s; Place QIAprep column in a clean 1.5 ml tube; Add 50 µl elution buffer (EB) to center of column, let stand for 3 min; Centrifuge: @ 13000rpm, 1min
************ Text for lab journal: *****************
Plasmid DNA Purification
experiment date: §§§ time: §§§
name of investigator: §§§
plasmid: name: §§§ number: §§§; production date: §§§; origin: §§§
ligation: new plasmid name: §§§; ligation date: §§§; origin: §§§; (optional vector name: §§§ insert name: §§§)
competent cells: strain name: §§§ date: §§§; origin: §§§; (optional competency: §§§ clones/µg)
Centrifuged cells: §§§ rpm, §§§ min
Discarded supernatant
Resuspended pellet in Buffer P1: §§§ µl
Transfered to a microcentrifuge tube
Addition of Buffer P2: §§§ µl; Mixed by inverting the tube §§§ times
Addition of Buffer N3: §§§ µl; Mixed by inverting the tube §§§ times
Centrifuged: §§§ min @ §§§ rpm
Applied supernatant to spin column
Centrifuged: @ §§§ rpm, §§§ s; Discarded flow-through
Washed spin column by adding: §§§ ml Buffer PB
Centrifuged: @ §§§ rpm, §§§ s;Discarded flow-through;
Washed spin column by adding: §§§ ml Buffer PE
Centrifuged: @ §§§ rpm, §§§ s
Discarded flow-through
Centrifuged: @ §§§ rpm, §§§ s
Placed column in a clean §§§ ml tube
Addition of §§§ µl elution buffer (EB) to center of column, let stand for §§§ min
Centrifuged: @ §§§ rpm, §§§ s
Measured DNA concentration per Nanodrop
************ End Text for lab journal: Plasmid Purification *****************
PCR Purification
Place one purification column for each sample in a collection tube
Add 500 µl of Capture Buffer to the column
Transfer the DNA solution (up to 100 µl) to the column, if purifying a PCR sample, avoid transferring mineral oil to the column
Mix thoroughly by pipetting the sample up and down 4-6 times
Centrifuge in a microcentrifuge at full speed for 30 s
Discard flow trough
Add 500 µl of Wash Buffer to the column, centrifuge at full speed for 30 s
Discard collection tube, place column in a new 1.5 ml Eppi
Apply 50 µl of Elution Buffer directly to the top of the glas fiber matrix in the column
Incubate the column for 3 min at room temperature
Centrifuge for 1 min at full speed to recover the purified DNA
Gel extraction
Use QIAquick Gel Extraction with Microcentrifuge
Excise DNA fragment from agarose gel with a clean, sharp scalpel Weigh gel slice Add:3 volumes Buffer QG to 1 volume of gel (100 mg ~ 100 μl) Incubate: at 50°C for 10 min (or until gel slice has completely dissolved) Add: 1 gel volume of isopropanol to sample, mix Place a spin column in a provided 2 ml collection tube To bind DNA: apply the sample to the column Centrifuge: for 1 min at 13000rpm (max. volume you can add to column is 800 μl) For sample volumes of more than 800 μl: simply load and spin again Discard flow-through Add: 0.5 ml Buffer QG to QIAquick column Centrifuge: 1 min at 13000rpm,discard flow-trough Add: 0.75 ml Buffer PE to column, centrifuge 1 min at 13000rpm Discard flow-through Centrifuge: 1 min at 13000 rpm to let the column dry Place column 1.5 ml eppi To elute DNA: add 50 μl elution buffer (EB) to the center of the membrane, let stand for 3min Centrifuge the column: for 1 min at 1300rpm
Agarose Gel
1% Agarose gel:
- 50 ml TAE buffer
- 0,5 g Agarose
- 2,5 µl Ethidium bromide
SDS gel
use Hoefer Mighty Small unit.12.5 % SDS gels: 9 gels.
Separating gel:
H20 21,38 ml
30% Acrylamide 28,13 ml
1.5 M Tris HCl pH 8.8 15,75 ml
20 % SDS 337,5 µl
10% APS 675 µl
TEMED 45 µl
Collecting gel:
H2O 30,60 ml
30 % Acrylamide 7,65 ml
1 M Tris HCl pH 6.8 5.63 ml
20 % SDS 225 µl
10 % APS 450 µl
TEMED 45 µl
Leammli buffer
Laemmli buffer (5x) 100ml:
1M Tris-HCl pH 6.8 30ml
SDS 10g
Glycerol 50ml
DTT 500mM
Bromphenol blue 250mg
SDS-PAGE
Dissolve samples in Leammli buffer Heat for 5 min at 95°C Spin down insoluble material (5min at full speed) Load samples into the sample wells Run gel at ca. 100 V for 10 min until all of the sample has migrated into the stacking gel, then at 150V until the dye front has reached the very bottom of the gel
PCR
Reagents
water fill up to 50 µl
buffer(10x)5 µl
dNTP's(10 mM each)1 µl
primer fwd 1 µl
primer rev 1 µl
template DNA 100 ng
polymerase 1 µl
Steps
1)94°C 4 min
2)94°C 1 min
3)annealing temp.30 sec
4)72°C 1 min
5)RETURN TO STEP 2, 29 cycles
6)72°C 10 min
7)6°C hold
Protein expression
Inoculate 100 ml LB+antibiotic and let grow over night at 37°C. Measure OD on the next day and inoculate 1000 ml (in a 2l baffled shake flask (Schikanekolben))of LB+antibiotic so that the OD of the 1000ml+overnight culture is about 0.1-0.2 For example: If the OD of the overnight culture is 5.0 --> Add 20-40 ml of the overnight culture--> new OD will be 0.1-0.2 Let grow until the OD reached 0.5-0.8 Induce with 1 mM IPTG Let grow at specific temperature (for Ago: 20°C) in shaker Centrifuge culture in 50 ml Falcon tubes at 4000rpm, 20 min, 4°C
Measurement of protein concentration
use UV/vis photometer press spectra measurement take black cuvette (not for phages)follow settings under measure -> parameters:
- general: accumulation --> 3
- control: baseline correction --> partial baseline, light source --> auto
- info: register sample name
measure from 220-350 nm
first set baseline (baseline button) with blank (e.g. dialyse buffer)
add protein solution to buffer (normally so that you have a 1:10 dilution)
--> e.g. take 135ul buffer as blank, then add 15ul of protein
clean cuvette with Helmanex, then use deionised water, at last use air pressure
His-tag column
Protein Purification of Aa with 2 ml Ni-NTA-Column
Sonificate cell extract 6x for 1 min
Filtering 44 µm + 22 µm
Run 10 ml of 20% ethanol trough column, pump speed 8-9x
Water 15 ml
Buffer 15 min
Coat samples: pump speed 5x ; collect flowtrough in 10 ml tubes
Add 2x 4 ml Buffer to rinse
Wash with 10 ml 30 mM Imidazol, collect in 10 ml fractions
Elution with 250 mM Imidazol: 12 fractions à 1.5 ml collect in small tubes
Wash column with 10 ml 250 mM Imidazol, 15 ml Water, 10 ml of 20% ethanol
Measure elution fractions: OD280 -> Analyze on SDS-gel
Overnight Culture/ Competent Cells
chemical
Inoculate 3ml LB+antibiotic, Incubate at 37°C over night Inoculate 200 ml LB+antibiotic with 2ml from the overnight culture
Let grow at 37°C on shaker, until OD is between 0,2-0,5 Let cell suspension stand on ice for 10-20 min, centrifuge at 3000 rpm; 20 min; 4°C Discard supernatant Resuspend pellet in 4ml cold CaCl2 solution (80mM) and add further 16 ml of CaCl2 solution Leave on ice for 30 min and Centrifuge 3000rpm; 20 min; 4°C Discard supernatant Resuspend pellet in 2.5 ml CaCl2/Glycerol (4:1), aliquot in Eppendorf tubes (100µl each), quick-freeze in liquid nitrogen, store at -80°C
electrical
Inoculate 3ml LB+antibiotic Incubate at 37°C over night Inoculate 200 ml LB+antibiotic with 2ml from the overnight culture Let grow at 37°C on shaker, until OD is between 0,2-0,5 Let cell culture stand on ice for 15 min Centrifuge at 2500g,4°C,10min Discard supernatant Resuspend pellet in 25 ml H2O Leave 15 min on ice Centrifuge at 2500g,4°C,10min Discard supernatant Resuspend pellet in 25 ml H2O again Leave 15 min on ice Centrifuge at 2500g, 4°C, 10min Discard supernatant Resuspend pellet in 25 ml 10% DMSO Leave 5 min on ice Centrifuge at 2500g, 4°C, 10min Discard supernatant Resuspend pellet in 5 ml 10% DMSO Leave 5 min on ice Centrifuge at 2500g, 4°C, 10min Discard supernatant Resuspend pellet in 1 ml 10% DMSO, aliquot in Eppendorf tubes (100µl each), quick-freeze in liquid nitrogen, store at -80°C
Western blot
Prepare SDS- gels
Transfer to PVDF (Polyvinylidenfluoride) membrane using iBlot Dry Blotting System
Put the PVDF membrane in blocking solution (TBS + 3% NFDM- Non-Fat-Dry-Milk) for 1h at room temperature (rocking platform)
Incubate with Antibody for 1h at room temperature (rocking platform)
Wash with TBST (Tris Buffered Saline + Tween 0.05%+ 3% NFDM) for
-1x 5 min
-2x 10 min on rocking platform
Detect Antibodies with Pierce ECL Western Blotting substrate
LB/2YT medium
LB:
10g/l Bacto Trypton
5g/l Yeast Extract
5g/l NaCl
2YT:
16g/l Bacto tryptone
10 g/l Bacto yeast extract
5g/l NaCl
LB-Agar and casting plates
Weigh: 6g agar agar Fill up with 400ml LB and treat with autoclave If the medium solidified: warm it up at 400 Watt for 15min Cool down to 60°C before you add antibiotic Before casting you can add antibiotic ( ratio 1:1000; for example 500ml LB + 500µl antibiotic) Mix softly you shouldnt have bubbles in your LB! Cast plates in the castingroom, its enough to cover just the bottom of the plates Sign the plates with date, iGEM and a green stripe on the lid when you added Ampicillin Close them and stack them in the castingroom, let them solidify under UV-light When they are soldified put them into the cooling room
Nano drop
start the PC, then start NP-1000 (Photometer) V3.7.1 username: FreiGEM, password: FreiGem1! choose type of measurement nucleic acid or protein put 1.5 µl water on the measure spot and click "OK" choose for nucleic acid if you want to measure DNA or RNA clean the spot and put again 1.5 µl water on it; click "Blank" measure your samples 1.5 µl each, clean between measurements after the measurement, clean with water use "Print Report" to get a printout for the labjournal
OD measurement
Over night cultures dilute 1:10 with LB before measurement Fresh started cultures can be measured without dilution Start the PC, start the program "Spectra Manager" and choose "Fixed wavelength measurement" on the right side of the program window Set the wavelength to 600 nm Measure a blank with LB and set "Autozero" Measure the Absorption
Buffersolution for anion exchanger
pH should be 1-2 pH- value over pi of protein Prepare a large amount to set up pH- value: 1. Prepare stock solution with 25mM Tris (set up pH- value) 2. Separate in: Running buffer (for 5l stock solution 3l Running buffer) 25mM NaCl , 25mM Tris
Elution buffer (for 5l stock solution 1l Elution buffer) 0,5M NaCl , 25mM Tris
Regeneration buffer (for 5l stock solution 0.5l Regeneration buffer)1M NaCl , 25mM Tris
Digestion
Example:
natural FokI with ssDNA from m13phage and 2x 40bp Oligos put the following components into a small eppi:
5µl MgCl2 (5mM)
5µl TrisHCl buffer(10mM)
5µl M13 phage ssDNA (c=186,05ng/µl)
5µl Oligo1 (must be same ammount of molecules as DNA)
5µl Oligo2 (must be same ammount of molecules as DNA)
25µl dest H2O
- - -
50µl
put the eppi into the pcr machine, use the program Origami1 this will take about 2h. Heating and cooling steps are required for propper annealing of Oligo and ssDNA
digestion:
2µl FokI
3µl buffer 4(required for FokI, if you use a differennt enzyme look on NEB table)
25µl of the DNA solution(see above)
37°C room, shake for 2h
Agarosegel:use 50ml TAE-buffer, 0,5g Agarose, 500µl Cybergreen II(this stains ssDNA) look up dilution
Ligation
Combine 50 ng of vector with a 3-fold molar excess of insert. Adjust volume to 10 μl with dH2O.
Add 10 μl of 2X Quick Ligation Buffer and mix.
Add 1μl of Quick T4 DNA Ligase and mix thoroughly.
Centrifuge briefly and incubate at room temperature (25°C) for 5 minutes.
Chill on ice, then transform or store at -20°C.
Do not heat inactivate. Heat inactivation dramatically reduces transformation efficiency.
Strep-Tag Manual
After protein extract has entered the strep tactin matrix, wash column 5x with 1 CV (column volume) buffer W
Elute recombinant protein by the addition of 6 times 0.5 CV buffer E
Regenerate the column by the addition of 3x 5 CV buffer R
Equilibrate the column by the addition of 2x 4 CV buffer W prior to the next purification run
Store the column at 4°C overlaid with 2ml buffer W or R
Agarose-Formaldehyde Electrophoresis
10 x MOPS buffer:
0.2M MOPS (morpholinopropanesulphonic acid)
50mM sodium acetate
5mM EDTA
The buffer is adjusted to pH 7.0 with 1M NaOH and sterilised by autoclaving.
RNA denaturing buffer:
10ml 100% deionized formamide
3.5ml 40% Formaldehyde (Take care: Formaldehyde is extremely toxic)
1.5ml 10 x MOPS buffer
Formamide is deionized by stirring 100ml with approximately 20g of Amberlite MB3 (or MB1) ion exchange resin for 15 minutes.
Agarose is prepared by melting the required amount of agarose in distilled water, cooling to approximately 60°C (hand hot) and adding 40% formaldehyde and 10 x MOPS to give 2.2M formaldehyde and 1 x MOPS, respectively. Example: For 50ml of a 1% agarose gel, melt 0.5g agarose in 37ml H2O, cool to hand hot, add 5ml 10 x MOPS buffer and 8.75ml 40% formaldehyde
Antibiotic stock solution preparation
Take Ampicillin-powder Mix with autoclaved H2O in ratio 100 mg/ml or 100mg/ml in 70% EtOH(100%EtOH+H20 sterile filter(22 from Rotilabo)) Press the mixture trough 100 ml syringe with a sterile filter (22 from Rotilabo) Freeze (at -20°C)in 1ml stocks
DNA Sequencing
Measure DNA/Plasmid concentration per Nanodrop Required DNA concentration and volume details from GATC website:
Concentration:
Plasmides DNA: 30-100ng/µl
PCR products: 10-50ng/µl
Customer primer: 10pmol/µl
Volume:DNA samples & primers in separate tubes: 30µl each (sufficient for 8 reactions)
Please send the DNA dissolved in water. The solution must not contain any EDTA!
Templates/primers in 1.5ml reaction tubes.
