Team:Stony Brook/Notebook/Cancer-W9

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Week 9: 8/22 - 8/28

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8/22

PCR of new IDT Shipments

AmilCP

Contents	ul
Q5 MasterMix	12.5
H2O	9
10 uM F Primer	1.25
10 uM R Primer	1.25
Template	1

IDT #1

Contents	ul
Q5 MasterMix	12.5
H2O	6.5
10 uM F Primer	2.5
10 uM R Primer	2.5
Template	1

IDT #2

Contents	ul
Q5 MasterMix	12.5
H2O	6.5
10 uM F Primer	2.5
10 uM R Primer	2.5
Template	1

PCR Settings: 25 Cycles

Phase	Temperature (°C)	Time (sec)
Initial Denaturation	98	30
Denaturation	98	10
Annealing	65	30
Extension	72	120
Final Extension	72	120

Digest of BioBricks

J23119 - 225.8ng

Content	Experimental ul	Control ul
Cutsmart	5	5
EcoRI-HF	0.5	X
SpeI	0.5	X
DNA	44.3	2.2

B0015 - 201.5ng

Content	Experimental ul	Control ul
Cutsmart	5	5
XbaI	0.5	X
PstI	0.5	X
DNA	49.6	2.5

E0040 - 179.3ng

Content	Experimental ul	Control ul
Cutsmart	5	5
EcoRI-HF	0.5	X
SpeI	0.5	X
DNA	55.8	2.8

LB Prep

J23119

• 8ml LB
• 2ml chloramphenicol

K592009

• 8ml LB
• 2ml chloramphenicol

E0040

• 8ml LB
• 2ml Ampicillin

B0015

• 8ml LB
• 2ml chloramphenicol

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8/23

PCR of amilCP, #1 and #2 with 67° annealing temperature

• Ran a gel afterwards

Lanes:

1. Ladder
2. AmilCP
3. #1
4. #2

Gel of 10ug overnight BB digest

Lanes:

1. Ladder
2. J23119
3. J23119 Control
4. B0015
5. B0015 Control
6. E0040
7. E0040 Control

Competent Cells (TSS)

50ml TSS

Content	Amount
PEG3350	5g
1M MgCL2	1.5 ml
DMSO	2.5ml
LB	Fill to 50ml

• Tested pH to be ~6.5

New PCR of Toggle #1 and amil CP

• 69°C annealing temperature
• Extension length - 2.5 mins
• Ran for 30 Cycles

Ran a gel

Lanes:

1. amil CP
2. #4
3. Ladder

Gel of Digested BB Parts

Lanes:

1. Ladder
2. J23119 #1
3. J23119 #2
4. B0015 #1
5. B0015 #2
6. E0040 #1
7. E0040 #2

Nanodrops of BioBricks and PCR purifications

Content	Concentration (ng/ul)	260/280
R592009	234.3	1.92
J23119	93.0	1.95
B0015	229.4	2.00
E0040	287.5	1.95
PiggyBac	274.0	1.89
Toggle #2	9.6	2.45

Digests

• Re-do of the the digest from 8/21

PCR of Amil CP and #1

• 67°C annealing temperature for 15 seconds
• 60 second extension

25ul PCR of Toggle #2

• 68°C annealing temperature
• Primer concentrations at 100-500 ng/ul

Gel of BioBrick Digests

Gel #1

Lanes:

1. Ladder
2. B0015
3. B0015
4. B0015 Control
5. J12119
6. J23119
7. J23119 Control

Gel #2

Lanes:

1. Ladder
2. K592009
3. K592009
4. K592009
5. K592009 Control
6. E0040
7. E0040
8. E0040 Control

PCR of Toggle #2 - 30x

Contents	ul
Q5 MasterMix	12.5
H2O	5.5
10 uM F Primer	1.0
10 uM R Primer	1.0
Template	5

Contents	ul
Q5 MasterMix	12.5
H2O	3.5
10 uM F Primer	2.0
10 uM R Primer	2.0
Template	5

Gel

Lanes:

1. Ladder
2. Toggle #2 - 1ul primer
3. Toggle #2 - 2ul primer

Toggle #1 Trials

PCR of Toggle #1

• Ran 6 trials. The first started with 0.5 ul of each of the F and R primers.
• Each trial increased by 0.5 until we got to 2.5ul of primers
• Ran a 0.25ul primer trial
• Used 12.5ul of Q5 MasterMix and 5ul of template

Toggle #1 Gel

Lanes:

1. Ladder
2. 0.25
3. 0.5
4. 1.0
5. 1.5
6. 2.0
7. 2.5

New PCR of Toggle #1 - Same settings as earlier Toggle #1

Contents	ul
Q5 MasterMix	12.5
H2O	X
5 uM F Primer	4
5 uM R Primer	4
Template	4.5

• Also ran PCRs with 2.0 and 2.5 ul of primers

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8/24

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8/25

Nanodrop of PCR products

PCR Products

Content	Concentration (ng/ul)	260/280
2.0 #1	43.7	1.87
2.5 #1	46.9	1.91
4.0 #1	54.7	1.94
1.0 #2	13.8	1.86
2.0 #2	31.1	2.18

BB Gel Extracts

Content	Concentration (ng/ul)	260/280
J23119	3.8	2.68
E0040	13.2	2.19
B0015	8.7	1.45
K592009	41.3	1.89

PCR of Toggle #2

Contents	ul
Q5 MasterMix	12.5
H2O	6.5
5 uM F Primer	0.5
5 uM R Primer	0.5
Template	5

• Also ran PCRs with 1.5, 2.5 and 3.5 ul of primers

BioBrick Digests

K592009 - 234.3ng/ul

Content	Experimental ul	25 ul Control
H2O	X	20.37
Cutsmart	15.8	2.5
EcoRI-HF	1.0	X
SpeI	1.0	X
DNA	140	2.13

J23119 - 93.0ng/ul

Content	Experimental ul	25 ul Control
H2O	X	17.12
Cutsmart	15.8	2.5
EcoRI-HF	1.0	X
SpeI	1.0	X
DNA	140	5.38

B0015 - 229.4ng/ul

Content	Experimental ul	25 ul Control
H2O	X	20.32
Cutsmart	15.8	2.5
XbaI	1.0	X
PstI	1.0	X
DNA	140	2.13

E0040 - 287.5 ng/ul

Content	Experimental ul	25 ul Control
H2O	X	20.76
Cutsmart	15.8	2.5
EcoRI-HF	1.0	X
SpeI	1.0	X
DNA	140	1.74

PCR Purification and Nanodrop of Toggle 2

Tube #	Concentration (ng/ul)	260/280
#2	12.3	1.36
#3	27.3	1.49

Digests of Toggles 1 and 2

Toggle 1

Content	Experimental ul
H2O	12.36
2.1 Buffer	2.5
EcoRI-HF	0.5
SpeI	0.5
DNA	9.14

Toggle 2

Content	Experimental ul
H2O	12.36
2.1 Buffer	2.5
EcoRI-HF	0.5
SpeI	0.5
DNA	9.14

Ligation

Content	ul
2.1 Buffer	5
B5A	0.5
Backbone	4ul
EcoRI-HF	0.5
PstI	0.5
T4 Mastermix	4ul
H2O	18.5

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8/26

Met with Dr. Zainul about our project

• Test would have to be administered in a lab
• Only on an at risk population (family history, heavy alcohol consumption, etc.)
• Would need to get the protocol down and license to sell it to a lab
• Not all private practices have centrifuges, so doing the test with a visually detectable indicator won't work
• Not all private practices have UV lights, so the draw of this being fast may not be as great as we thought
• Maybe consider light chromatography
• If this can be done in a private practice, we'll need a sort of stick test, similar to the glucose and protein tests for urine
• Can consider coupling GPC-1 binding to secretion of a selected product that can bind to an external indicator that can be poured into the solution, like phenolphthalein

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8/28

PCR of new IDT Shipments

AmilCP

Contents	ul
Q5 MasterMix	12.5
H2O	7
5 uM F Primer	0.25
5 uM R Primer	0.25
100 ug/uL Template	5

• The 5 uM F primers and the 5 uM R primers went up in increments of 0.5 uL until we reached 2.5 uL
• The concentration of water started at 7 uL and went down by 0.5 uL for each successive PCR until we reached 2.5 uL

PCR Settings Template:

Phase	Temperature (°C)	Time (sec)
Initial Denaturation	98	30s
Denaturation	98	10s
Annealing	67	15s
Extension	72	60s
Final Extension	72	120s