Team:PKU Beijing/Notebook/Assembly/Haoqian Zhang

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Notebook > Assembly > Haoqian Zhang's Note

2009.10.1

Positively transform 1-9C plasmid, intending to get pSB3T5 plasmid backbone.

One 5ml culture of LB medium and antibiotic (Tetracycline, 50ng/ml) was inoculated with a single positive colony from a LB agar plate. Cultures were grown in tubes for 14 hrs at 37°C with shaking at 70 rpm.

2009.10.2

Miniprep of the fresh culture containing 1-9C plasmid.

Digest 1-9C plasmid and T7p + RBS + CI plasmid with XbaI and PstI Double digestion system:

1.5μLXbaI
1.5μLPstI
2μL10×M buffer
10μL1-9C plasmid (5μL for T7p + RBS + CI plasmid)
5μLddH2O (5μL for T7p + RBS + CI plasmid)
20μLTotal

Electrophoresis the digested samples, and extract the 1-9C plasmid backbone and T7p + RBS + CI insert.

2009.10.3

Ligate the T7p + RBS + CI insert to 1-9C plasmid backbone. System:

3μLinsert
1μLT7p vector
1μL10× Ligase buffer
1μLLigase
4μLddH2O
10μLTotal

Transform the ligation product, and plate on agar plate.

2009.10.4

Pick 3 colonies for each RBS, 18 colonies in total, incubated in LB medium with Tetracycline, 50ng/ml for 14 hrs.

Miniprep these 18 culture samples.

Digest the plasmids with XbaI and PstI to detect the successful ligation. Double digestion system:

1.5μLXbaI
1.5μLPstI
2μL10×M buffer
10μLsample plasmid
5μLddH2O (5μL for T7p + RBS + CI plasmid)
20μLTotal

Electrophoresis the digested samples detect the successful ligation. The result showed that all the colonies did come from successful ligation.



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