Team:Cambridge/LabBook/Week3

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Contents

Tuesday

1. Experiment: Streaking out of bacterial cultures (Peter & Anja)

On LB agar plates:

  • TOP10
  • MG1655
  • W3110 hns 93-1
  • BW25113

On LB agar + kan plates:

  • BW25113 Δhns::kan
  • BW25113 ΔtraA::kan

Incubated at 37°C overnight

Wednesday

all strains grew with individual colonies.

2. Experiment: Set up overnight culture of TOP10 (Emily & Anja)

Inoculated single bacterial colony (ATP10) in 12ml LB, incubated at 37°C with shaking (180 rpm) overnight.

Thursday

Inoculated 400ml LB with 5ml TOP10 overnight culture, incubate at 37°C with shaking (180rpm) put on at 11:40am

OD600 measurements:

Time OD600 (1) OD600 (2)
12:20pm -0.026 -0.032
2:30pm 0.437 0.530

3. Preparation: CCMB80 Buffer (Volume500ml)

Arrows label dilutions to the indicated concentration

  • KOAC 1M ---> 10mM 5ml
  • CaCl2.2H20 1M ---> 80mM 40ml
  • MnCl2.4H20 1M ---> 20mM 10ml
  • MnCl2.6H20 1M ---> 10mM 5ml
  • glycerol --->10%
  • sterile H20 390ml
  • prepare in flow hood
  • sterile filter
  • store at 4°C

4. Experiment: Set up overnight cultures of TOP10 (Will & Anja)

Inoculated single bacterial (TOP10) colony in 5ml SOB, incubated at rtp with shaking overnight

Friday

5.Experiment: Preparing chemicall competent cells (Emily, Bill & Anja)

(followed protocol for 'TOP10 chemically competent cells' from OpenWetWare)

Inoculated 800ml SOB with 3ml of TOP10 overnight culture (grown from single colony), incubated at 37°C with shaking (180rpm)

Put on at 9.45am

OD600 measurements:

Time OD600 (teaching lab) OD600 (Jim's lab)
10:50am 0.008 -0.002
11:30am 0.013 0.000
12:00pm 0.023 0.024
12:35pm 0.032 0.034
01:00pm 0.040 0.046
01:32pm 0.052 0.063
02:00pm 0.073 0.090
02:35pm 0.106 0.140
03:00pm 0.137 0.174
03:25pm 0.179 0.230
03:38pm 0.203 0.256
03:55pm 0.225 0.313

Cooled cells and CCMB80 buffer in ice bath for around 20min. Aliquoted 600ml culture in 12x50ml centrifuge tubes (pointed bottom). Centrifuged at 3000g at 4°C for 10min. Discarded supernatatent. Gently resuspended cell pellet in 20ml ice cold CCMB80 buffer per tube. Incubated on ice for 20min. Centrifuget at 3000g at 4°C for 10min. Pooled Pairs of tubes together. 12 tubes ---> 6tubes. Discarded supernatant. Resuspended pellet in 5ml ice for 20min. Aliquoted 150x200μl into Eppendorf tubes. Stored at -80°C.

Saturday & Sunday

6. Experiment: Measuring competency of TOP10 (Fernan)

TOP10 coopmetent cells (cc) taken out of -80°C freezer and put on ice. Check for thawing ater 5minutes (leave on ice). Cut 1ml pipette tips with scissors sterilised in ethanol and flamed with a Bunsen burner. Transfer ~50μl of TOP10 cc into 1.5ml Eppendorf tube with cut pipette tip. (will have to judge ~50μl by eye). Added 1μl of pUC19 (standard control plasmid) at 10-5μg/μl. Held on ice for 30min. Heat shockede for 60s at 42°C (waterbath). Put on ice for ~2min. Added 250μl pre-warmed (in 37°C incubator) SOC. Incubated at 37°C for 1h with rotation (for this purpose stick Eppendorf tubes in 12ml falcon tubes and put tape over the top). Plated 20μl on LB agar paltes with Amp (with blue shaped spreader). Grow colonies overnight at 37°C.

  • Yielded ~150 cells/plate
  • transformation efficiency (no. of transformed cells (colonies) generated by 1μg of plasmid DNA)
  • volume of cells * colonies on a plate / mass of DNA transformed
  • 15 * 150 * 105 = 2.25x108/ μgDNA
  • volume=50μl cells + 1μl plasmid + 250μl SOC, 301μl of which 2Oμl were plated, giving dilution factor of 15
  • mass of DNA is 10-5 μg


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