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

Ligate Toggle 2 into Plasmid

Content	ul
Ligase Buffer	2
Toggle pieces	10 (500 ng/ul)
Plasmid	2
H2O	5
Ligase	1

Phosphatase of linearized pSBK3

Amount	ul
Digestion of plasmid	10
Buffer	2
Phosphatase	1
H2O	7

Ligate into Plasmid treated with Phosphatase

Amount	ul
Plasmid	5
Toggle pieces	10 (500 ng/uL)
Buffer	2
H2O	2
Ligase	1

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8/31

Digest of IDT CR-1 SBU and Vector

CR-1 SBU

Content	Experimental ul	Control ul
H2O	18	20
Cutsmart	5.0	5.0
EcoRI-HF	1.0	X
XhoI	1.0	X
DNA	25.0	25.0

pEP352GAP

Content	Experimental ul	Control ul
H2O	7.0	8.0
Cutsmart	1.0	1.0
EcoRI-HF	0.5	X
XhoI	0.5	X
DNA	1.0	1.0

Incubation

• Incubated overnight

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9/1

Deactivation of Digest with Phosphatase

PCR of Toggle #1

• Same setup as 8/30

Ligation of CR-1 into pEP352GAP

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9/2

PCR of Toggle 1

Contents	ul
Phire	10
H2O	8.0
5 uM F Primer	0.5
5 uM R Primer	0.5
100pg/ul DNA	1.0

• Also ran trials with 1.5, 2.5, 3.5 and 4.5 ul of both forward and reverse primers
• Adjusted H₂O accordingly

PCR Settings: 30 cycles

Phase	Temperature (°C)	Time (sec)
Initial Denaturation	98	30
Denaturation	98	10
Annealing	67	5
Extension	72	25
Final Extension	72	60

Another PCR of Toggle 1

Contents	ul
Phire	10
H2O	7.74
10 uM F Primer	0.63
10 uM R Primer	0.63
100pg/ul DNA	1.0

• Ran trials with 1.25 and 1.88 ul of forward and reverse primers
• H₂O for those trials were 6.5 and 5.24 ul respectively

PCR Settings: 35 cycles

Phase	Temperature (°C)	Time (sec)
Initial Denaturation	98	30
Denaturation	98	10
Annealing	68.5	5
Extension	72	20
Final Extension	72	60

PCR of Toggle 2

Contents	ul
Phire	10
H2O	7.74
10 uM F Primer	0.63
10 uM R Primer	0.63
100pg/ul DNA	1.0

• Ran trials with 1.25 and 1.88 ul of forward and reverse primers
• H₂O for those trials were 6.5 and 5.24 ul respectively

PCR Settings: 35 cycles

Phase	Temperature (°C)	Time (sec)
Initial Denaturation	98	30
Denaturation	98	10
Annealing	68.5	5
Extension	72	35
Final Extension	72	60

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9/3

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9/4