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:
| 1.5μL | XbaI
|
| 1.5μL | PstI
|
| 2μL | 10×M buffer
|
| 10μL | 1-9C plasmid (5μL for T7p + RBS + CI plasmid)
|
| 5μL | ddH2O (5μL for T7p + RBS + CI plasmid)
|
| 20μL | Total
|
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:
| 3μL | insert
|
| 1μL | T7p vector
|
| 1μL | 10× Ligase buffer
|
| 1μL | Ligase
|
| 4μL | ddH2O
|
| 10μL | Total
|
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:
| 1.5μL | XbaI
|
| 1.5μL | PstI
|
| 2μL | 10×M buffer
|
| 10μL | sample plasmid
|
| 5μL | ddH2O (5μL for T7p + RBS + CI plasmid)
|
| 20μL | Total
|
Electrophoresis the digested samples detect the successful ligation. The result showed that all the colonies did come from successful ligation.
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