Team:Cambridge/LabBook/Week3

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{{:Team:Cambridge/Templates/headerbar|colour=#386abc|title=Lab Book: Week 3}}
{{:Team:Cambridge/Templates/headerbar|colour=#386abc|title=Lab Book: Week 3}}
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==27.07.10==
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==Tuesday==
===1. Experiment: Streaking out of bacterial cultures (Peter & Anja)===
===1. Experiment: Streaking out of bacterial cultures (Peter & Anja)===
On LB agar plates:
On LB agar plates:
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*BW25113 ΔtraA::kan
*BW25113 ΔtraA::kan
Incubated at 37°C overnight
Incubated at 37°C overnight
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==28.07.10==
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==Wednesday==
all strains grew with individual colonies.  
all strains grew with individual colonies.  
===2. Experiment: Set up overnight culture of TOP10 (Emily & Anja)===
===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.
Inoculated single bacterial colony (ATP10) in 12ml LB, incubated at 37°C with shaking (180 rpm) overnight.
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==29.07.10==
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==Thursday==
Inoculated 400ml LB with 5ml TOP10 overnight culture, incubate at 37°C with shaking (180rpm) put on at 11:40am
Inoculated 400ml LB with 5ml TOP10 overnight culture, incubate at 37°C with shaking (180rpm) put on at 11:40am
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===4. Experiment: Set up overnight cultures of TOP10 (Will & Anja)===
===4. Experiment: Set up overnight cultures of TOP10 (Will & Anja)===
Inoculated single bacterial (TOP10) colony in 5ml SOB, incubated at rtp with shaking overnight
Inoculated single bacterial (TOP10) colony in 5ml SOB, incubated at rtp with shaking overnight
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==30.07.10==
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==Friday==
===5.Experiment: Preparing chemicall competent cells (Emily, Bill & Anja)===
===5.Experiment: Preparing chemicall competent cells (Emily, Bill & Anja)===
(followed protocol for 'TOP10 chemically competent cells' from OpenWetWare)
(followed protocol for 'TOP10 chemically competent cells' from OpenWetWare)
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Discarded supernatant. Resuspended pellet in 5ml ice for 20min. Aliquoted 150x200μl into Eppendorf tubes. Stored at -80°C.
Discarded supernatant. Resuspended pellet in 5ml ice for 20min. Aliquoted 150x200μl into Eppendorf tubes. Stored at -80°C.
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==31.07.10 & 01.08.10==
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==Saturday & Sunday==
===6. Experiment: Measuring competency of TOP10 (Fernan)===
===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).
TOP10 coopmetent cells (cc) taken out of -80°C freezer and put on ice. Check for thawing ater 5minutes (leave on ice).
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*mass of DNA is 10<sup>-5</sup> μg
*mass of DNA is 10<sup>-5</sup> μg
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==02.08.10==
 
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===7. Experiment: Transformation of TOP10 cc (Ben, Emily, Bill, Hannah, Will & Anja)===
 
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Top10 cc taken out of -80°C freezer and tawed on ice. 1ml pipette tip cut with scissors sterilised with ethanol and flamed. Using cut pipette tip ~50μl of TOP10cc were transferred to 1.5ml Eppendorf tubes (3x)
 
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#2μl of resuspended (in 10μl deionised water) BBa_J13002 (plasmiid with Tet<sup>R</sup> represed PoPs/RIPs generator) was added.
 
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#2μl of resuspended (in 10μl deionised water) BBa_I712019 (plasmid with firefly luciferase) was added
 
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#1.5μl of pHK724 (plasmid containing lux4 gene) was addded
 
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Ce]]s were held on ice for 30min. Heat shocked for 60s at 42°C (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 250μl pre-wardmed (in 37°C) SOC. Incubated at 37°C for Ph with rotation (Eppendorf tubes---> 12ml falcon tubes, tape over top). Plated 50micorl on pre-warmed (in 37°C incubator) LB agar plates with Amp (with blue L-shaped spreader). Grow colonies overnight at 37°2.
 
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===8.Experiment: Set up overnight culture of E.coli/pHK555 (Will & Anja)===
 
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Inoculated single bacterial colony (E.coli/pHK555) in 5ml LB in a 12ml falcon tube. Incubated at 37°C with rotation overnight.
 
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(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)
 
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===9.Experiment: Set up overnight cultures of W3110 hns 93-1, BW25113 Δhns::kan, W3110 hns-205::Tn10 Tet<sup>R</sup>, GM230 hns-205::Tn10 Tet<sup>R</sup> (Ben & Anja)===
 
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Inoculated single bacterial colonies in 5ml SOB (i.e. 4 different cultures), incubated at RT with shaking overnight.
 
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==03.08.10==
 
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===10. Experiment: Preparing chemically competent cells (W3110 hns 93-1, BW25113 Δhns::kan,W3110 hns-205::Tn10 Tet<sup>R</sup>, GM230 hns-205::Tn10 Tet<sup>R</sup> (Ben, Will, Paul, Bill, Emily, Hannah & Anja)===
 
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(followed protocol for 'TOP10 chemically competent cells cells' from OpenWetWare)
 
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Inoculated 0.5ml of the 4 bacterial strains (overnight cultures) in 85ml SOB each. Incubated at 37°C with shaking (180rpm). put on at 11:55am
 
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Bacterial Strains:
 
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{| class="wikitable"
 
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|-
 
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! ID
 
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! Type
 
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|-
 
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| <font color="black">(1)</font>
 
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| GM230 hns-205::Tn10 Tet<sup>R</sup>
 
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|-
 
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| <font color="red">(2)</font>
 
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| BW25113 Δhns::kan
 
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|-
 
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| <font color="blue">(3)</font>
 
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| W3110 hns 93-1
 
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|-
 
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| <font color="green">(4)</font>
 
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| W3110 hns-205::Tn10 Tet<sup>R</sup>
 
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|}
 
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OD<sub>600</sub> measurements:
 
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{| class="wikitable"
 
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|-
 
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! Time
 
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! OD<sub>600</sub> <font color="black">(1)</font>
 
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! OD<sub>600</sub> <font color="red">(2)</font>
 
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! OD<sub>600</sub> <font color="blue">(3)</font>
 
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! OD<sub>600</sub> <font color="green">(4)</font>
 
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|-
 
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| 1:00pm
 
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| 0.019
 
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| -0.008
 
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| -0.020
 
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| 0.049
 
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|-
 
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| 2:00pm
 
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| 0.034
 
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| 0.027
 
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| 0.001
 
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| 0.124
 
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|-
 
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| 3:00pm
 
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| 0.059
 
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| 0.114
 
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| 0.049
 
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| 0.268
 
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|-
 
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| 3:25pm
 
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| 0.092
 
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| 0.235
 
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| 0.116
 
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| -
 
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|-
 
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| 3:40pm
 
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| -
 
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| 0.297
 
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| -
 
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| -
 
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|-
 
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| 4:00pm
 
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| 0.159
 
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| -
 
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| 0.267
 
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| -
 
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|-
 
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| 4:25pm
 
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| 0.203
 
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| -
 
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| -
 
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| -
 
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|-
 
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| 4:40pm
 
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| 0.256
 
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| -
 
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| -
 
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| -
 
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|}
 
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Cooled cells and (newly prepared) CCMB80 Buffer in ice bath for ~20 minutes. <font color="green">(4)</font> was on ice for over an hour. <font color="red">(2)</font> was on ice for ~40 minutes. <font color="blue">(3)</font> was on ice for ~40 minutes.
 
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2 x 30-35ml of each of the  four 85ml cultures were poured into 50ml falcon tubes. Centrifuged at 3000g at 4°C for 10 min. Discarded supernatant. Resuspended cell pellet in 20ml tube CCMB80 buffer (ice cold). On ice for 20 min. Centrifuged at 3000g at 4°C for 10 min. Discarded supernatant. Resuspended cell pellet in 5ml tube CCMB80 buffer. Incubated on ice for 20 min. Aliquoted 200μl portions into Eppendorf tubes colour-coded according to the above listed colour labels. Stored at -80°C
 
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===11. Experiment: Streaking out of bacterial cultures (<i>E. coli</i>/pHK555) (Paul & Anja)===
 
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On LB agar plates with Chloranphenicol: <i>E. coli</i>/pHK555 (from both overnight cultures) on two individual plates incubated at 37°C overnight.
 
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===12. Experiment: Set up overnight cultures (TOP10 with BBa-J13002, TOP10 with BBa_I712019) (Ben & Anja)===
 
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Results from transformations on 02/08/10
 
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* Colonies grew for TOP10 cells transformed with BBa_J13002 (promoter + rbs) and those transformed with BBa_I712019 (firefly luciferase) :-)
 
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* no cells grew for untransformed TOP10 cells plated on LB agar + Amp plates (neg control)
 
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* small (but plenty) colonies grew for TOP10 cells transformed with pHK724, these were left in the incubator for another 24h, and then put at 4°C
 
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Inoculated single bacterial (TOP10 with BBa-J13002 and TOP10 with BBa_I712019 individually) colony in 5ml LB, incubated at 37°C with shaking (180rpm) overnight
 

Revision as of 12:17, 9 August 2010

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