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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

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?

Recipes used in the lab

LB Broth

• 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:

• 0.4% carbon source, Glucose: 40 g
• 2mM MgSO4 → 0,2407 g / L 120,37
• 0.1mM CaCl2 → 0,0111 g / L
• 12,8g Na2HPO4.7H2O
• 3 g KH2PO4
• 0,5 g NaCl
• 1 g NH4Cl

In sterile H2O → 1000 mL

pH 7,4 , 4 x 250mL erlenmeyer, autoclave