Notebook : July 9

Summary :

For regulation system :

• extraction of BioBrick BBa_K1155000 in concentrated medium for further DNA sequencing.
• the terminator BBa_B0010 in PSB1A2 plasmidwas extracted form iGEM plate kit 2013 and was transformed into competent cells and then was cultured on solid medium
• the plasmid PSB3K3 was extracted form iGEM plate kit 2013 and transformed into competent cells and then was cultured on solid medium for confirmation

For sensor system:

• A series of digestion, ligation were performed for BioBrick BphR2, BphR1, BphA1

Lab work

DNA purification

See protocol DNA purification Briobrick promoter BphR1, BphR2, BphA1 were purified.

Restriction digestion

We had 5 samples for digestion: 3 extracts, plasmid PSB1C3 and Plasmid PSB1K3(extracted from plate kit 2013 6F ).

For the plasmid (PSB1C3 and PSB1K3):

• Plasmid : 4µl
• (orange): 0.8µl
• EcoR I: 0.5µl
• PST I:0.5µl
• H2O:2.2µl
• Total:8µl

For the DNA extract:

• DNA: 15µl
• Buffer(orange): 3µl
• EcoR I: 0.75µl
• PST I:0.75µl
• H2O:10.5µl
• Total:30µl

After the digestion, superfluous enzyme was removed by Ethanol precipitation method. Then we suspended them with 10µl H2O.

Quantification

Ligation

Common way for ligation.

• Plasmid : 2µl
• Bph : 2µl
• Buffer : 2µl
• T4 ligase : 12µl
• H2O : about 7µl
• Total : 20µl

Transformation

See protocol transformation terminator BBa_B0010 in PSB1A2 plasmid and plasmid PSB3K3was transformed into competent cells and then was cultured on solid medium.