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Team:NTNU Trondheim/Protocols
From 2011.igem.org
Protocols
Resuspending DNA from registry-parts:
- Poke a hole in foil of corresponding well
- Resuspend DNA in 10 µL dH2O (pipette up and down a few times)
- Wait 5 minutes.
- Transfer the resuspended DNA to pCR tube and store in -20C.
Transforming competent cells:
- Thaw competent cells on ice
- Mix with 2 µL plasmid DNA
- Incubate on ice 30 minutes
- Heat-shock cells 45 seconds in 42C water-bath.
- Incubate 5 minutes on ice
- Add 200 µL SOC
- Incubate the Eppendorf tubes in Erlenmeyer flask 37C shaking incubator for 2 hours
- Plate 50 µl and 200 µl of the transformation onto the dishes, and spread.
- Incubate ON in 37C
Isolating plasmids:
- Inoculate one colony in 3 mL LB with appropriate antibiotic. (IE 1,5µL amp stock in 3 mL LB)
- Grow in shaking incubator 30C ON
- Isolate plasmid using [http://www.ebiotrade.com/buyf/productsf/Promega/tb225.pdf SV Miniprep procedure]
- Store in -20C
Gel Extraction:
- Using QIAquick Gel Extraction Kit [http://molecool.wustl.edu/krolllab/Kroll_Lab_Protocols/Molecular%20Biology%20protocols/Cloning%20protocols%20folder/Gel%20extraction-Qiagen.pdf protocol]
- eluating with dH20
Restriction Digest:
- Add 500ng of DNA to be digested, and adjust with dH20 for a total volume of 42.5ul.
- Add 5ul of NEBuffer 2 to the tube.
- Add 0.5µl of BSA to the tube.
- Add 1µl of your first enzyme.
- Add 1µl of your second enzyme.
- There should be a total volume of 50ul. Mix well and spin down.
- Incubate the restriction digest at 37C for 30min, and then 80C for 20min to heat kill the enzymes.
Ligation:
- After digestion, separation, purification.
- Add 11ul of dH20
- Add 2ul from each sample you will be ligating (destination plasmid, and part)
- Add 2ul of T4 DNA Ligase Reaction Buffer
- Add 1ul of T4 DNA Ligase
- Mix well, and spin down.
- Incubate for 30min at 16C and 20min at 80C to heat kill.
- Use 2ul of ligation to transform into competent cells.
Alternatively:
- After gel extraction:
- Add 1 µL T4 DNA Ligase Reaction Buffer
- ca 6:1 molar ratio of insert to vector (~10ng vector)
- Add (8.5 - vector and insert volume)μl
- 0.5 μL T4 Ligase
PCR
PCR mix:
- Work on ice
- 0.5 µL DNA
- 5 µL 10x PCR buffer 2 (w/MgCl2)
- 0.5 µL 10 mM dNTPs
- 1 µL fwd primer
- 1 µL rev primer
- 0.5 µL polymerase
- 41.5 µL H20
PCR program:
- Temperature of lid: 103°C
- Heat cycles:
- Initial denaturation: 94°C for 2 min
- Denaturing: 94°C for 30 s
- Annealing: 58°C for 30 s
- Elongation: 72°C for 60 s
- Repeat 2-4 34 times
- Final elongation: 72C 7 min
- Cooling: 4 C HOLD
Testing of construct
Test 1 Nullmedium:
- Inoculate in 5 mL ON
- Dillute 4 x 1:100 in LB, grow to OD 0,5
- Pellet, resuspend 2x in 0,9% NaCl, 37C, 2x in LB
- Grow 3 hrs?
Test 2 Amino acid deprived:
- Inoculate in 5 mL ON
- Dillute 4 x 1:100 in LB, grow to OD 0,5
- Pellet, resuspend 2x in no amino acid media, 37C, 2x in LB
- Grow 3 hrs?
Test 3 Temperature:
- Inoculate in 5 mL ON
- Dillute 6 x 1:100 in LB, grow to OD 0,5
- Pellet, resuspend in LB, Incubate 2 x 25C, 2 x 37C, 2x 40C ?
- Grow 3 hrs?
TSS competent cells preparation:
- Make TSS
- Inoculate 5 ml LB (w/o antibiotic) from company stock stored in –80C and let grow o/n.
- Inoculate 30 ml LB (w/o antibiotic) with 300 μL from the o/n culture. (Alternative: Inoculate 3 ml for 5 hrs and dump into 30ml culture.)
- Monitor OD until it reaches 0.4-0.5.
- When OD reaches 0.4 remove from shaker and put on ice.
- Pellet the cells in 50ml falcon (or autoclaved tube) tube at 6000g, 4C for 10 minutes.
- Decant liquid and place cells back on ice.
- Resuspend cells in 3.0ml TSS, pipet up and down and vortex. (resuspend in 10% original volume of TSS. ie. 30ml culture, 3 ml TSS)
- Pipet 250 μL cells into each prelabeled and precooled microfuge tube
- Store at –80C
Making blunt ends on DNA with sticky ends:
T4 DNA polymerase was used (Fills in 3' overhang and removes 5' overhang.
- Digested DNA was cooled on ice, 2 µl 2 mM dNTP and 1 µl T4-DNA polymerase was added.
- The mix was incubated at 16 C for 12 min.
- Inactivate enzyme and isolate fragment by using PCR purification kit.
Recipes used in the lab
LB medium
- Bacto-tryptone: 10 g/L
- Yeast extract: 5 g/L
- NaCl 5 g/L
Adjust pH to 7,4 with NaOH, autoclave.
LA
- Dissolve LB
- Add agar, 15 g/L
Adjust pH to 7,4 with NaOH, autoclave.
SOC:
- Bacto-tryptone: 20 g/L
- Yeast extract: 5 g/L
- NaCl 0,5 g/L
- 10 mL 250 mM KCl (25mM) → 0,186 g / 10mL H20
- 5 mL 2M MgCl2 (10mM) → 0,952 g / 5mL
Autoclave, cool, then add:
- 20 mL 1M sterile-filtered glucose (20mM)
Ampicillin-plates:
Use stock 200 mg/mL. → 100µg/mL in liquid agar. I.E. 250 µL stock in 500 mL agar. Add when cool enough to pour.
Spectinomycin-plates:
Use stock 100 mg/mL. → 100µg/mL in liquid agar. I.E. 500 µL stock in 500 mL agar. Add when cool enough to pour.
Ampicillin-IPTG plates:
Ampicillin 100 µg / mL (500 µL amp stock (200 mg/mL) in 500 mL agar) IPTG 0,1 mM ( 63,5 µL IPTG stock (0,8 M))
M9 minimal medium
Make M9 salts
- To make M9 Salts aliquot 800ml H2O and add
- 33.93g Na2HPO4 16,7 g
- 15g KH2PO4 7,5g
- 2.5g NaCl 1,25g
- 5.0g NH4Cl 2,5g
- Stir until dissolved
- Adjust to 1000ml with distilled H2O
- Sterilize by autoclaving
- Measure ~700ml of distilled H2O (sterile)
- Add 200ml of M9 salts
- Add 2ml of 1M MgSO4 (sterile)
- Add 20 ml of 20% glucose (or other carbon source)
- Add 100ul of 1M CaCl2 (sterile)
- Adjust to 1000ml with distilled H2O
Transfection Storage Solution (TSS)
50 ml TSS:
- 10 % Polyethylene glycol 5 g
- 5 % Dimethyl Sulfoxide (DMSO) 2,5 ml
- 20 mM MgCl2 1 ml
- Bring to 50 ml with autoclaved LB
- Stir until dissolved
- Filter through 0.22 μM syringe filter
- Autoclave centrifuge tubes, flasks and bottle/beaker for TSS
- Store at -20 C
