Team:NTNU Trondheim/Protocols

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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 42.5 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 37C 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 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?

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

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