Team:Hamburg/Experiments

Experiments & Protocols

Stocks and working supplies

Chloramphenicol Stock:

P4 mg/ml preparation of Chloramphenicol Stock (3 in EtOH)

  1. dissolve 34 mg of Chloramphenicol in 1 ml 100% ethanol
  2. filter through a 0.22 μm filter to sterilize
  3. use at 1:1000 dilution in LB or LB-Agar
  4. comment: prepare bigger stock next time (10 ml). It’s not very exact working with 34 mg CAmp
  5. mark as CAmp (top) and CAmp / dd.mm.yy / 34 mg/ml (side) in Antibiotics
  6. store at -20 °C

LB-Agar Plates (+ Chloramphenicol/ + Kanamycin):

  1. Heat 250 ml LB-Agar
  2. Cool down to ca. 50 °C
  3. Add 250 μl Chloramphenicol/ 12.5 μg Kanamycin (50 µg/ml) / 25 µL Ampicillin (80 µg/mL)
  4. Preparation of 20 plates under sterile conditions
  5. Store at 4 °C, mark as LB CAmp/Kan/Amp Plates iGEM, dd.mm.yyyy

Annealing Buffer:

  • 0.5 M EDTA (provided by Dr. Shinichiro Komaki)
  • 5 M NaCl (provided by Dr. Shinichiro Komaki)
  • 1 M Tris (self-made:made in 10 mL ddH2O, pH 8.04)

for 10 mL:

  • EDTA 20 µL
  • Tris 100 µL
  • NaCl 100 µL
  • + H2O → 10 mL

SOC medium:

(instruction “Roche, Lab FAQ”, by Dagmar Stang)

  • Bacto-Tryptone 2.000 g
  • Bacto-Hefe-Extract 0.500 g
  • NaCl 0.050 g
  • KCl 0.019 g
  • MgCl2 * (H2O)6 0.203 g (without (H2O)6 → 0.095 g)
  • Glucose 0.036 g

pH 7.0 with pH-Meter

→ 10 mL (fridge)

TB PIPES Buffer:

for 100 mL:

  • KCl 1.865 g
  • CaCl x 2 H20 0.22 g
  • 0.5 M PIPES 2 mL
  • pH 6.7
  • MgCl2 x 2H2O 0.889 g
  • ddH2O → 100 ml

Working instructions

Competent Cells:

Reagents:

  • Cells
  • LB plate
  • LB medium
  • TB-PIPES buffer
  • Liquid nitrogen

Day 1:

  1. Flame the metal inoculating loop
  2. Scrape off a portion from the top of the frozen (DH5-alpha) cells
  3. Streak it onto the LB plate
  4. Put the stock back to -80 °C immediately
  5. Leave the plates for 5 minutes and place them upside down in the 37 °C incubator overnight

Day 2:

  1. Pick a single colony into 3 mL of LB medium
  2. Inoculate the culture over night at 37 °C with shaking at 200 rpm
  3. Transfer 0.5 mL of the overnight preculture into 50 mL LB medium (1-2 L Erlenmeyer flask)
  4. Let the culture grow to OD600 = 0.6 at 18 °C with shaking at 200 rpm
  5. Transfer the culture into a 50 mL Falcon tube and leave for 10 min on ice
  6. Spin down cells at 3500 rpm for 8 min at 4 °C
  7. Resuspend in 1/3 volume TB-PIPES buffer (17 mL for 50 mL original culture)
  8. Leave for 10 min on ice
  9. Spin down cells at 3500 rpm for 8 min at 4 °C
  10. Resuspend in 1/12.5 volume ( 4 mL for 50 mL original culture)
  11. Add 7% DMSO (sterile)
  12. Leave for 10 min on ice
  13. Aliquot 50 µL in pre-chilled Eppendorf tubes and freeze by immersion in liquid nitrogen
  14. Store at −80 ° C

Transformation Efficiency ‘Competent Cell Test Kit’:

(iGEM Help: Transformation Efficiency Kit)

  1. spin down DNA (0.5, 5, 10, 20 & 50 pg/µL RFP Construct BBa_J04450, pSB1C3) from Competent Cell Test Kit ( 30 sec, 10.000 rpm)
  2. thaw competent cells on ice, label one 2 ml tube for each concentration + pre-chill
  3. 1 µl of DNA in each tube, respectively
  4. add 50 µl of competent cells to each tube, incubate for 30 min on ice
  5. pre-heat (42 °C), heat shock for 1 min
  6. incubate for 5 min on ice
  7. add 200 µl SOC media, incubate for 2 h at 37 °C
  8. pipet 20 µl on each plate (triplets), respectively
  9. incubate overnight at 37 °C
  10. count colonies, calculate transformation efficiency

Annealing:

  1. get the amount of DNA from the delivery note
  2. dilute both oligos to 100 μM
  3. anneal in water bath at 94 °C for 10 min
  4. cool to room temperature in water bath over 60 min
  5. store solution at 4 °C in fridge

Control of Annealing:

  • in agarose gel (1%)
  • 1 µL loading dye
  • 9 µL sample (or appropriate amount & add H20 up to 9 µL)

Make agarose gel:

  • 0.5 g Agarose
  • 50 mL TAE-buffer (gel room)
  1. Heat in microwave until agarose is dissolved
  2. Transfer into gel slide, tip pipet into EtBr, then into gel
  3. Cool for 30 minutes

Restriction:

20 µL pSB-1C3 (500 ng)

1 µL EcoRI

1 µL PstI

3 µL FD Buffer

5 µL ddH2O

20 minutes at 37 °C, following clean up with kit

Backbone dephosphorylation:

  • 10 µL pSB1C3 (cut)
  • 1 µL FastAP
  • 4 µL FastAP Buffer

→ 37 °C for 30 minutes

→ purify with PCR clean up kit

Ligation:

1 µL pSB-1C3 vector (23 ng)

1 µL /1.5 µL GroEl-Insert (3.5 ng) / miRNA-Insert (3.3 ng)

2 µL T4 Buffer

1 µL T4 Ligase

5 µL ddH20 ( 6 µL for control)

15 min at room temperature, then 10 min at 65 °C

Transformation:

  • 10 µL ligation product
  • 50 µL competent cells
  1. 30 min on ice
  2. 1 min Heatshock at 42 °C
  3. 15 min on ice
  4. add 200 µL SOC-Medium
  5. 2 h at 37 °C on rotator
  6. plate out on appropriate plates (C-Amp/ Kan)
  7. incubate over night at 37 °C

Pick colony:

  • Inoculate each picked colony into 5 mL of liquid LB-(CAmp/ respective antibioticum)-medium, respectively
  • Tubes in the 37 °C incubation room, on rotator!!

Minipreparation:

  • use Geneaid Mini Plasmid Preparation Kit
  • store samples in freezer

Glycerol stocks:

from liquid culture

  • 500 µL (50% Glycerol, 50% H2O)
  • 500 µL BW29655 or DH5a
  • → -80°C

Preparation for sequencing:

  • Primer concentration: 100 µM→ Concentration needed: 5 µM (dilute at 1:20)
  • take 50 nm of sample mix with 5 µL of ( 5 µM primer, only forward)


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