IGEM:PKU Beijing/2009/Notebook/AND Gate 1/Output/2009/07/02/Zhangs

Miniprep 10 samples
CI + 1-23L samples: 1-5
CI + 1-4H samples: 1-5
Measure the concentration of these 10 samples.
CI + 1-23L sample 1 400ng/uL
sample 2 385ng/uL
sample 3 900ng/uL
sample 4 875ng/uL
sample 5 515ng/uL
CI + 1-4H sample 1 250ng/uL
sample 2 1000ng/uL
sample 3 250ng/uL
sample 4 370ng/uL
sample 5 215ng/uL
Electrophoresis: Agarose 2%
Identify the ligation of CI+1-23L/1-4H by electrophoresis
Result (from left to right)
Marker, 1-23L vector control, CI+1-23L①~⑤ plasmids, 1-4H vector control, CI+1-4H①~⑤ plasmids

Causing the CI+1-4H samples are not very good, we do electrophoresis again: Agarose 2%.

Put the ligated 1-4H + CI plasmids into 1 EP tubes. Conc = 395ng/uL.

Miniprep for 13 samples RBS plasmid:
Nine 2009 iGEM parts samples 1-2I, 1-2G, 1-2M, 1-5J, 1-5N, 1-1J, 1-1H, 1-2K, 1-11N
Four 2008 iGEM parts samples 1004-1C, 1004-2G, 1004-3C, 1004-4C
The resistances are all Amp+

Double digestion
12 RBS samples: 9 2009 iGEM parts samples 1-2I, 1-2G, 1-2M, 1-5J, 1-5N, 1-1J, 1-1H, 1-2K, 1-11N
3 2008 iGEM parts samples 1004-2G, 1004-3C, 1004-4C
System for back vector:

10μL	plasmid
2μL	10×H buffer
1.5μL	PstI
1.5μL	SpeI
5μL	ddH2O
20μL	Total

2 CI-1-23L samples & 2 CI-1-4H samples
Systems for back insert:

10μL	plasmid
2μL	10×M buffer
1.5μL	XbaI
1.5μL	PstI
5μL	ddH2O
20μL	Total

Water bathing for 4 hours.
Electrophoresis the digestion product.
Sample loading order: CI + 1-4H sample 2, 3, 4; CI + 1-23L sample 1, 2; Marker.

The digestion of CI + 1-4H 4 failed.
Electrophoresis
Sample loading order: Marker, 1-11N control (plasmid), 1-11N digested, 1-4H4 control (plasmid), CI + 1-4H4

CI + 1-4H sample failed to be digested again.