Team:Stony Brook/Notebook/Cancer-W10

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Week 10: 8/29 - 9/4

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8/29

AmilCP BioBrick Digests

Content	Experimental ul	Control ul
H2O	46.5	47.0
Cutsmart	5.0	5.0
XbaI	0.5	X
DNA	2.0	2.0

PCR of Toggle 1

Contents	ul
Phire	10
H2O	8
5 uM F Primer	1.5
5 uM R Primer	1.5
100pg/ul Construct	1.0

• Ran other trials with 2.5, 3.5, 4.5 and 6.5 ul of Forward and Reverse Primers
• Water ul was adjusted to compensate: 6, 4, 2 and 0 ul respectively

Phase	Temperature (°C)	Time (sec)
Initial Denaturation	98	30
Denaturation	98	5
Annealing	68	5
Extension	72	45
Final Extension	72	60

PCR of Toggle 2

Contents	ul
Phire	10
H2O	8
5 uM F Primer	1.5
5 uM R Primer	1.5
100pg/ul Construct	1.0

• Ran other trials with 2.5, 3.5, 4.5 and 6.5 ul of Forward and Reverse Primers
• Water ul was adjusted to compensate: 6, 4, 2 and 0 ul respectively

Phase	Temperature (°C)	Time (sec)
Initial Denaturation	98	30
Denaturation	98	5
Annealing	68	5
Extension	72	45
Final Extension	72	60

Digests of Toggle parts

Toggle 1 Digest

Content	Experimental ul	Control ul
H2O	24	25
Cutsmart	5.0	5.0
SpeI	1.0	X
DNA	25.0	25.0

Toggle 1 Digest

Content	Experimental ul	Control ul
H2O	24	25
Cutsmart	5.0	5.0
XbaI	1.0	X
DNA	25.0	25.0

Linearized pSB1C7 Digest

Content	Experimental ul	Control ul
H2O	16.5	18.5
Cutsmart	2.5	2.5
SpeI-HF	1.0	X
XbaI	1.0	X
DNA	4.0	4.0

Ligation of Toggle parts

Content	ul
10X T4 Buffer	2.0
Toggle 1	5.0
Toggle 2	10.0
H2O	2.0
Ligase	1.0

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8/30

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8//31

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9/1

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9/2

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9/3

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9/4