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Team:Cambridge/LabBook/Week3
From 2010.igem.org
Lab Book
27.07.10
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
28.07.10
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.
29.07.10
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
30.07.10
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.
31.07.10 & 01.08.10
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
02.08.10
7. Experiment: Transformation of TOP10 cc (Ben, Emily, Bill, Hannah, Will & Anja)
Top10 cc taken out of -80degreesC freezer and tawed on ice. 1ml pipette tip cut with scissors sterilised with ethanol and flamed. Using cut pipette tip ~50microl of TOP10cc were transferred to 1.5ml Eppendorf tubes (3x)
- 2microl of resuspended (in 10microl deionised water) BBa_J13002 (plasmiid with TetR represed PoPs/RIPs generator) was added.
- 2microl of resuspended (in 10microl deionised water) BBa_I712019 (plasmid with firefly luciferase) was added
- 1.5microl of pHK724 (plasmid containing lux4 gene) was addded
Ce]]s were held on ice for 30min. Heat shocked for 60s at 42degreesC (waterbath). As a control TOP10cc that had not been transformed with anything (no plasmid DNA added) were subjected to same treatment (as cells to be transformed). Put on ice for ~2min. Added 250microl pre-wardmed (in 37degreesC) SOC. Incubated at 37degreesC for Ph with rotation (Eppendorf tubes---> 12ml falcon tubes, tape over top). Plated 50micorl on pre-warmed (in 37degreesC incubator) LB agar plates with Amp (with blue L-shaped spreader). Grow colonies overnight at 37degrees2.
8.Experiment: Set up overnight culture of E.coli/pHK555 (Will & Anja)
Inoculated single bacterial colony (E.coli/pHK555) in 5ml LB in a 12ml falcon tube. Incubated at 37degreesC with rotation overnight. (Since it was difficult to see the colony sent from Jim Slock, we picked twice from where we anticipated colony to be and set up 2x5ml LB overnight cultures)
9.Experiment: Set up overnight cultures of W3110 hns 93-1, BW25113 deltahns::kan, W3110 hns-205::Tn10 TetR, GM230 hns-205::Tn10 TetR (Ben & Anja)
Inoculated single bacterial colonies in 5ml SOB (i.e. 4 different cultures), incubated at RT with shaking overnight.
03.08.10
10. Experiment: Preparing chemically competent cells (W3110 hns 93-1, BW25113 deltahns::kan,W3110 hns-205::Tn10 TetR, GM230 hns-205::Tn10 TetR (Ben, Will, Paul, Bill, Emily, Hannah & Anja)
(followed protocol for 'TOP10 chemically competent cells cells' from OpenWetWare) Inoculated 0.5ml of the 4 bacterial strains (overnight cultures) in 85ml SOB each. Incubated at 37degreesC with shaking (180rpm). put on at 11:55am
