IGEM:Groningen/Notebook/iGEM 2011/2011/09/08

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8-9-11




Plasmid Prep of PBAD-cI-LVA, PBAD-cI-LVA-DT, PBAD-LasR-LVA-DT and PBAD-RBS-GFP-DT in vector
Nanodrop ND1000 results: DNA in sample, between 35 and 75 ng/μl
Still no stickers for sending them to sequence the plasmid :(.
Use the PBAD-cI-LVA vector isolated this morning for digestion

Cloning PBAD-cI-LVA-DT in two ways
Calculations were made with the Ligation calculator based on DNA concentrations according to the nanodrop
Digestion:
DT
2μl insert
1μl XbaI
1μl PstI
2μl FD buffer
14μl MQ water

Both PBAD-cI-LVA vectors:
2.5μl vector
1μl SpeI
1μl PstI
3μl FD buffer
1μl FastAP
21.5μl MQ

Incubate the samples for 1h at 37 °C.

DNA clean up with the High Pure PCR Purification Kit of Roche

Ligation:
PBADaraC-cI-LVA-DT
2.4μl DT
8.5μl vector RBS-GFP-DT
2μl T4 DNA ligase buffer
1μl T4 DNA ligase
6.1μl MQ water

Self ligation control:
8.5μl vector
2μl T4 DNA ligase buffer
1μl T4 DNA ligase
8.5μl MQ water

Incubate the samples for 30 to 40 minutes at roomtemperature

Transformation
Add to 40μl competent cells 10μl ligation mixture
Incubate for 30 min on ice
Heat shock: 45s at 42 degrees
Incubate cells on ice for 2 min
Add 1ml LB medium+ 25mM glucose
Incubate cells for 1h/1.5h at 37 degrees
Spin the cells down, resuspend them in 100μl LB medium and plate 90μl and 10μl out on the plates
Put the plates in the stove at 37 degrees overnight

Preparing overnight flasks for FACS. Also made reservation for tomorrow 9-9-11 FACS from 12h-18h
10ml of M9 and 1ml of 2% casaminoacids. Inoculate PBAD-RBS-GFP-DT colony 4 and 6
Read the article: A fast, robust and tunable synthetic gene oscillator
Jesse Stricker1*, Scott Cookson1*, Matthew R. Bennett1,2*, William H. Mather1, Lev S. Tsimring2 & Jeff Hasty1,2
where they induce with 0.7% arabinose.
Make a stock of 10% arabinose:
10% w/w Arabinose Stock Solution:
Add 1g of Arabinose to 9ml of milliQ water in a small falcon tube. Filter sterilize with a 0.22μm filter.
Can be stored at room temperature. From: http://openwetware.org/wiki/Arabinose


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