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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 a PCR tube and store in at -20°C.

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 42°C water-bath.
• Incubate 5 minutes on ice
• Add 200 µL SOC
• Incubate the Eppendorf tubes in Erlenmeyer flask 37°C shaking incubator for 2 hours
• Plate 50 µl and 200 µl of the transformation onto the dishes, and spread.
• Incubate ON in 37°C

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 30°C ON
• Isolate plasmid using [http://www.ebiotrade.com/buyf/productsf/Promega/tb225.pdf SV Miniprep procedure]
• Store in -20°C

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 42.5 µl.
• Add 5µl 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 50 µl. Mix well and spin down.
• Incubate the restriction digest at 37°C for 30 min, and then 80°C for 20 min 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 16°C and 20min at 80°C to heat kill.
• Use 2µl 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, 37°C, 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, 37°C, 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 25°C, 2 x 37°C, 2x 40°C ?
• Grow 3 hrs?

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