IGEM:PKU Beijing/2009/Notebook/AND Gate 1/Core/2009/07/21/Shan Shen

00:40
Digest SupD and terminator plasmids.
System of SupD:

Total	20μL
Plasmids	6μL
EcoR1	1μL
Spe1	1μL
Buffer	2μL
ddH2O	10μL

System of terminator 1-23L:

Total 20μL

Plasmids 6μL

EcoR1 1μL

Xba1 1μL

Buffer 2μL

ddH2O 10μL

1:00
Start to digest.

10:00
Electrophoresis to test the digestion products.
The order and the amount of the samples: marker 5μL, SupD digestion, 1-23L plasmids, 1-23L digestion.

Results:
The digestions are successful.

11:10
Recycle the fragments of SupD.

13:10
Ligation of the vectors of 1-23L and inserts of SupD.
System of ligation: 10μL

	Control system	1:7 system
Vectors	1μL	1μL
Inserts	0μL	7μL
Buffer	1μL	1μL
Ligase	1μL	1μL
ddH2O	7μL	0μL

13:10
Ligation starts.

14:21
Miniprep bi-stable 1,2,3,SupD1 and 1-9H.
Concentrations:

Number of the plasmids	Concentration(ng/μL)
Bi-stable 1	160
Bi-stable 2	200
Bi-stable 3	250
SupD1	250
1-9H	110

17:20
Transform the ligation products of SupD+1-23L backbone.