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Missouri_Rolla

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30 October 2017

Ryan Baumann

Start: 5:00PM

Cloning efficiency testing of new Universal Acceptor Plasmid

Purpose: To test the cloning efficiency of the Universal Acceptor Plasmid after deletion of the unexpected 10 bp region via site directed mutagenesis PCR

Protocol:

Golden gate assemblies were performed to insert TNTR3 gblock into our universal acceptor plasmid. Various molar ratios of TNTR3: Universal acceptor plasmid were tested. Molar ratios of 1:2, 1:1, 2:1 (recommended ratio), 4:1, and 8:1 were tested for the new plasmid. A 2:1 ratio assembly was performed using our original UAP. A negative control assembly was performed with no inserts added. Golden gate assemblies contained the following: 1:2 Ratio

Component	Volume
TNTR3 (17.64 ng)
New UAP (100ng)	1 μL
10X Ligase Buffer	2 μL
Ligase	1 μL
BsmBI	1 μL
MilliQ H2O	up to 20μL

1:1 ratio

Component	Volume
TNTR3 (35.28 ng)
New UAP (100ng)	1 μL
10X Ligase Buffer	2 μL
Ligase	1 μL
BsmBI	1 μL
MilliQ H2O	up to 20μL

2:1 Ratio

Component	Volume
TNTR3 (70.56 ng)
New UAP (100ng)	1 μL
10X Ligase Buffer	2 μL
Ligase	1 μL
BsmBI	1 μL
MilliQ H2O	up to 20μL

4:1 Ratio

Component	Volume
TNTR3 (141.12 ng)
New UAP (100ng)	1 μL
10X Ligase Buffer	2 μL
Ligase	1 μL
BsmBI	1 μL
MilliQ H2O	up to 20μL

8:1 Ratio

Component	Volume
TNTR3 (282.24 ng)
New UAP (100ng)	1 μL
10X Ligase Buffer	2 μL
Ligase	1 μL
BsmBI	1 μL
MilliQ H2O	up to 20μL

2:1 Ratio with original UAP

Component	Volume
TNTR3 (70.56 ng)
Original UAP (100ng)	1 μL
10X Ligase Buffer	2 μL
Ligase	1 μL
BsmBI	1 μL
MilliQ H2O	up to 20μL

Negative Control

Component	Volume
TNTR3	None
UAP	None
10X Ligase Buffer	2 μL
Ligase	1 μL
BsmBI	1 μL
MilliQ H2O	up to 20μL

All samples were run with the following thermocycler conditions: – 20 seconds @ 37°C, – (3 minutes @ 37°C, 4 minutes @ 16°C) X26 – 5 minutes @ 50°C – 5 minutes @ 80°C – 5 minutes @ 16°C Following assembly, 1 uL of each sample was transformed on to Chloramphenicol Blue-White Screening plates following Intact Genomics ig 5-Alpha chemically competent cell High efficiency Transformation protocol. Dillutions of 20% and 5% were plated and spread using sterile glass beads. A positive control transformation was performed using 1 uL of original UAP. Colonies were incubated at 37C for 30 hours. Blue and white colonies were counted on all plates.

Stop: 1:45AM

Results:

See project page

Next:

Boston!

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25 October 2017

Ryan Baumann

Start: 3

Blunt end Ligation of 10/22/17 PCR1 and transformation

Purpose: Ligation of UAP back to itself after removal of 10bp sequence.

Protocol:

Blunt end ligation was performed uisng NEB T4 DNA ligase protocol. 2 uL of 10/22/17 OJM PCR 1 was ligated in 20 uL reaction. Reaction was conducted at room temperature for 2 hours. After Ligation, 2 uL of 10/25/17 L1 was transformed using Intact Genomics ig 5-Alpha chemically competent cell High efficiency Transformation protocol. 50% and 5% dilutions were plated onto Chloramphenicol plate.

Notes:

Check colony growth

Stop: 5:00

Products:

Label	Source	Description
10/25/17 L1	10/22/17 PCR1	UAP Ligated back to itself following removal of 10bp fragment

Next:

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23 October 2017

Ryan Baumann

Start: 8:45PM

Colony PCR's of 10/18/2017 Transformations of PhoB and TNTr3, gels of Golden Gate Round two PCR's and 10/23/17 PCR 1 and 2

Purpose: To check assembly of golden gate round two assemblies and test OJM1 and OJM2 primers.

Protocol:

18 Colony PCRs were conducted using Taq 2x Master Mix protocol. 9 Colonies from PhoB and TNTR3 transformation plates were used as template. PCR 1-9 - TNTR3 PCR 10-18 PhoR Three 1% agarose gels were prepared with 2 uL of ethidium bromide Gel 1:

Lane(s)	Component
1	2 Log purple DNA ladder
2	TNTR3 PCR1
3	2 Log purple DNA ladder
4-10	TNTR3 PCR 2-3 and 5-9
11	2 Log purple DNA ladder
12-20	PhoR PCR 1-9

Gel 2:

Lane(s)	Component
1	2 Log purple DNA ladder
2-8	10/23/17 cPCR's 1-7 (Q92X31)
13	2 Log purple DNA ladder
14-20	10/23/17 cPCR's 8-14 (PhoB)
23	10/23/17 OJM PCR1

Gel 3:

Lane(s)	Component
1	2 Log Purple DNA Ladder
2-8	10/23/17 cPCR 15-21 (O22527)
11	2 Log Purple DNA Ladder
12-18	10/23/17 cPCR 22-28 (Q8LDU4)

Notes:

Stop: 10:30

Next:

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23 October 2017

Jeremy Mesa

Start: 5:00 pm

Purpose: To perform a PCR with mutagenesis primers in order to delete a short base pair sequence from plasmid.

Protocol:

Contents of PCR tubes:

	Q5	oJM1	oJM2	UAP	Milli-Q
PCR 1	12.5 μL	1.25 μL	1.25 μL	1 μL	4 μL
PCR 2	12.5 μL	1.25 μL	1.25 μL	0 μL	5 μL

PCR 2 was a negative control. Thermal Cycler Program:

	Temp (°C)	Time	Cycles
Step 1	98	30 sec	0
Step 2	98	5 sec	24
Step 3	65	20 sec	24
Step 4	72	75 sec	24
Step 5	72	2 min	0
Step 6	4	Infinite

Stop: 5:45

Next:

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23 October 2017

Ryan Baumann, Jessica Brooks, Tiffany Kuhnert, Lucas Dyer

Start: 4:15pm

Colony PCR of 10/23/2017 Q92x31 5%, Q22527 5% , PhoB 50% , and Q8LD44 5% , golden gate round 2 transformation plates

Purpose: To confirm assembly of title parts with their promoters and terminators

Protocol:

28 Colony PCRs were conducted using Taq 2x Master Mix protocol. 7 colonies from each plate were run along with a RFP Construct postitive control and milliQ water negative control. 1-7 Q92X31 8-14 PhoB 15-21 O22527 22-28 Q8LDU4 Reaction Set Up:

Component	Volume (μ)
Taq 2x Master Mix	7.5
VR	0.3
VF2	0.3
MilliQ	6.9
Colony1-34

Thermocycler Conditions:

	Temp. (C)	Time	Repeats
Step 1	95	5 min	0
Step 2	95	30 sec	35
Step 3	51	30 sec	35
Step 4	68	3 min	35
Step 5	68	5 min	0
Step 6	4

Stop:

Next:

Run Gels of all the colony PCRs

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18 October 2017

Ryan Baumann

Start: 2:30PM

Plasmid mini preps of Q9LX31, Q8LDU4, and O22527 transformations. Golden gate assemblies of PhoB, PhoR, TNTR3, Q9LX31, Q8LDU4, and O22527 and transformation on to Kanamysin Blue white screening plates

Purpose: Purify degreening plasmids and assemble signal transduction pathway, periplasmic binding protein, and degreening parts into transcriptional units.

Protocol:

Minipreps were conducted using iGEM lab manual 2.5 kitless miniprep protocol. Samples were resuspended in 50 uL of TE buffer. DNA Concentration were calculated using Thermoscientic Nanodrop 1000. Nanodrop Results:

Sample	DNA Conc. (ng/μL)	260/280
10/17 MP3 (O22527)	122.1	2.11
10/17 MP4 (O22527)	364.9	1.86
10/17 MP5 (Q9LX31)	326.9	1.88
10/17 MP6 (Q9LX31)	280.2	1.91
10/18 MP1 (Q8LDU4)	76.7	1.93
10/18 MP2 (Q8LDU4)	59.3	1.90
10/18 MP3 (K1467101)	47.4	1.93
10/18 MP4 (K1467101)	100.3	1.93

Golden Gate Assemblies of PhoB (9/8/17 MP3), PhoR (9/13/17 MP1), TNTR3 (9/8/17 MP1), O22527 (10/17/17 MP4), Q9LX31 (10/17/17 MP5), Q8LDU4 (10/18/17 MP2) were performed following the NEB Golden Gate assembly kit protocol into golden braid alpha 1 acceptor plasmid (BBa_P10501). The plant pho promoter (9/8/17 MP2) was added to the degreening parts. BBa_K1467101 added to TNTR3, PhoR, and PhoB. BBa_K1618037 (9/13/17 MP3) was added to all parts. 2 uL of each golden gate assemblies were transformed using Intact Genomics ig 5-Alpha chemically competent cell protocol. 50% and 5% dilutions were plated onto Kanamysin Blue white screening plates.

Stop: 8:30PM

Results:

Check for colonies

Products:

Label	Source	Description
10/17 MP3	O22527	O22527 in the UAP
10/17 MP4	O22527	O22527 in the UAP
10/17 MP5	Q9LX31	Q9LX31 in the UAP
10/17 MP6	Q9LX31	Q9LX31 in the UAP
10/18 MP1	Q8LDU4	Q8LDU4 in the UAP
10/18 MP2	Q8LDU4	Q8LDU4 in the UAP
10/18 MP3	K1467101	K1467101 in the UAP
10/18 MP4	K1467101	K1467101 in the UAP
10/18 GG1	9/8/17 MP3	PhoB with BBa_K1467101 Promoter and BBa_K1618037 Terminator
10/18 GG2	9/13/17 MP1	PhoR with BBa_K1467101 Promoter and BBa_K1618037 Terminator
10/18 GG3	9/8/17 MP1	TNTR3 with BBa_K1467101 Promoter and BBa_K1618037 Terminator
10/18 GG4	10/17 MP4	O22527 with Plant Pho Promoter and BBa_K1618037 Terminator
10/18 GG5	10/17 MP5	Q9LX31 with Plant Pho Promoter and BBa_K1618037 Terminator
10/18 GG6	10/18/17 MP2	Q8LDU4 with Plant Pho Promoter and BBa_K1618037 Terminator

Next:

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17 Oct. 2017

Jeremy Mesa

Start: 2:30

Purpose: To run PCR with mutagenesis primers and a gel following to visualize the results of the reaction.

Protocol:

PCR: Contents of the tube, 20 μL total

Tube	Q5	oJM1	oJM2	UAP	Milli-Q
PCR 1	10 μL	1 μL	1 μL	1 μL	7 μL

Thermal Cycler Program:

	Temp. (°C)	Time	Repeats
Step 1	98	30 sec	0
Step 2	98	10 sec	30
Step 3	65	20 sec	30
Step 4	72	2 min	30
Step 5	72	2 min	0
Step 6	4	Infinite	0

1% Agarose gel was prepared with 3 μL Ethidium Bromide.

Lane 1	Ladder	20 μL
Lane 4	PCR 1	15 μL

Notes:

Program name on thermal cycler is saved as PCR10-6.

Stop: 4:00

Results:

PCR lane of gel was found to have 4 bands at about 2500bp, 1500bp, 600bp, and less than 100bp. The lane was streaked behind the largest base pair band.

Next:

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10 15 2017

Lynell Cunningham, Luke Dyer, Tiffany Kuhnert

Start: 3:00

Colony PCR and Gel of 9/30/17 Golden gate assemblies of degreening parts

Purpose: To confirm assembly of Q9LX31, Q8LDU4, and O22527 into the universal acceptor plasmid.

Protocol:

17 Colony Pcr's were conducted using Taq 2x Master mix protocol. 5 Colonies from each 9/30/17 golden gate reaction were run along with a RFP Construct postitive control and milliQ water negative control. Reaction setup:

Component	Volume
Taq 2X Master Mix	10 μL
VR	.4 uL
VF2	.4 uL
MilliQ	9.2 uL
Colony 1-15

Thermocycler Conditions: 95C for 5 minutes, 35 Cycles of: (95C for 20 seconds, 66C for 30 sec, 68C for 1min), 68C for 5 minutes, and 4C hold All samples were run on a 1% agarose gel with the following setup: Lane 1 and 11: 2 log purple DNA ladder Lane 2-6 are Colonies 1-5 from Q8LDU4 Lanes 7,8,9,10,and 12 are Colonies 6-10 from O22527 Lanes 13-17 are Colonies 11-15 for Q9LX31 Lane 18 is a water negative control Lane 19 is the RFP Construct positive control After imaging, one colony from each golden gate transformation was grown in broth culture containing Chloramphenicol

Notes:

Stop: 10:00pm

Next:

Grow successfully assembled cultures, miniprep, and round two assemblies

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10 October 2017

Ryan Baumann

Start: 4:30PM

Gel of 9 October 2017 Golden Gate and Colony PCR's

Purpose: To confirm proper assembly of previous golden gate assemblies

Protocol:

A 1% agarose gel with 1.5 μL Ethidium Bromide and 2 Log Purple DNA Ladder.10 μL of samples were loaded into wells.

Well	Sample
1	2 Log purple DNA Ladder
2	1
3	2
4	3
5	4
6	5
7	6
8	7
9	8
10	9
11	2 Log Purple DNA Ladder
12	10
13	11
14	12
15	13
16	14
17	15
18	16
19	17
20	18
21	10/9/17 A1
22	10/9/17 A2
23	10/9/7 A3

Notes:

Stop: 6:00:00PM

Results:

Analyze Gel Results

Next:

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30 September 2017

Kent Gorday, Lynell Cunningham, Lucas Dyer, Jeremy Mesa

Start: 16:35

Diagnostic PCRs of round 2 assemblies and assembly of degreening gblocks into UAP

Purpose: To check addition of constitutive promoter and terminator to coding sequences, and assemble degreening coding sequences into Universal Acceptor Plasmid for submission.

Protocol:

Six PCRs were performed from round 2 assemblies:

9/30 A1-6

10 μL	2X Q5 Master Mix
7 μL	MilliQ Water
1 μL	Template Plasmid
1 μL	VF2
1 μL	VR
20 μL	Total Volume

Temperature (°C)	Time (sec)
98	30
98c	8
65c	22
72c	93
72	120
4	ꝏ

cycle X32

Three Golden Gate reactions into the Universal Acceptor Plasmid were performed:

9/30 GG1-3

4.9 μL	100 ng/μL Q9LX31 gblock
2.5 μL	UAP
2 μL	10X T4 DNA Ligase Buffer
1 μL	T4 DNA Ligase
1 μL	BsmBI
8.6 μL	MilliQ Water
15.1 μL	Total Volume

5.9 μL	100 ng/μL O22527 gblock
2.5 μL	UAP
2 μL	10X T4 DNA Ligase Buffer
1 μL	T4 DNA Ligase
1 μL	BsmBI
7.6 μL	MilliQ Water
14.1 μL	Total Volume

5.8 μL	100 ng/μL Q8LDU4 gblock
2.5 μL	UAP
2 μL	10X T4 DNA Ligase Buffer
1 μL	T4 DNA Ligase
1 μL	BsmBI
7.7 μL	MilliQ Water
14.2 μL	Total Volume

Temperature (°C)	Time (sec)
37	20
37c	180
16c	240
50	300
80	300
16	300

cycle X32

A 1.2% agarose gel was run with PCR products:

Well No.
3	Ladder
4	A1
5	Ladder
6	A2; Bad Load
7	A4
8	A3; Bad Load
9	A5
10	A6

Stop: 20:40

Results:

Products:

Label	Source	Description
9/30 A1	9/18 MP1	+ pro/ter amplified
9/30 A2	9/18 MP2	+ pro/ter amplified
9/30 A3	9/18 MP3	+ pro/ter amplified
9/30 A4	9/18 MP4	+ pro/ter amplified
9/30 A5	9/18 MP5	+ pro/ter amplified
9/30 A6	9/18 MP6	+ pro/ter amplified
9/30 GG1	Q9LX31 gblock	GUN4 CDS assembled into Universal Acceptor Plasmid
9/30 GG2	O22527 gblock	Chlorophyllase-1 CDS assembled into Universal Acceptor Plasmid
9/30 GG3	Q8LDU4 gblock	RCCR CDS assembled into Universal Acceptor Plasmid

Next:

Colony PCR and patch to a new plate a few white colonies from each round 2 assembly transformation.

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9-21-17

Ryan Baumann

Start: 1:30PM

PCR and Gel of 18 September 2017 minipreps (again)

Purpose: To identify if phoR, phoB, and TNTR3are properly assembled in transcriptional units with BBa_K1618037 and BBa_K1467101

Protocol:

Six 20 μL PCR's of 9/18/17 MP 1-6 were run using containing: Q5 HIgh Fidelity 2X Master Mix, 0.4 μL VR and VF2 (10 μM solutions), The following template concentrations, and MilliQ Water up to 20 μL.

Template	Volume
9/18/17 MP1	1 μL
9/18/17 MP2	1 μL
9/18/17 MP3	1 μL
9/18/17 MP4	1 μL
9/18/17 MP5	4 μL
9/18/17 MP6	5 μL

A positive control was run using a 1μL of RFP construct in psb1C3. A negative control was run using MilliQ water. Samples were run in the thermocycler in the following conditions: 98C for 30 seconds 32 cycles of: 98C for 8 seconds, 65C for 22 seconds, and 72C for 85 seconds 72C for 120 seconds 4C Hold All PCR's were run on a 1% agarose gel containing 1.5 μL of ethidium bromide

Well	Sample
1	2 Log Purple DNA Ladder
3	PCR1(MP1)
4	PCR2(MP2)
5	PCR3(MP3)
6	PCR4(MP4)
7	PCR5(MP5)
8	PCR6(MP6)
9	Positive Control
10	Negative Control

Notes:

Stop:

Results:

Continue to troubleshoot PCR conditions. No conclusive results from gel.

Next:

Redo PCR's again

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21 September 2017

Ryan Baumann

Start: 2:00 PM

PCR and Gel of 18 September 2017 minipreps

Purpose: To identify if phoR, phoB, and TNTR3are properly assembled in transcriptional units with BBa_K1618037 and BBa_K1467101

Protocol:

Six 20 μL PCR's of 9/18/17 MP 1-6 were run using containing: 300ng of template DNA Q5 High Fidelity 2X Master Mix, 0.2 μM VR and VF2 and water up to 20 μL A positive control was run using an RFP construct in psb1C3. A negative control was run using MilliQ water. Samples were run in the thermocycler in the following conditions: 98C for 30 seconds 32 cycles of: 98C for 8 seconds, 65C for 22 seconds, and 72C for 85 seconds 72C for 120 seconds 4C Hold All PCR's were run on a 1% agarose gel containing 1.5 μL of ethidium bromide

Well	Sample
1	2 Log Purple DNA Ladder
3	PCR1(MP1)
4	PCR2(MP2)
5	PCR3(MP3)
6	PCR4(MP4)
7	PCR5(MP5)
8	PCR6(MP6)
9	Positive Control
10	Negative Control

Notes:

Stop: 5:30PM

Next:

TBD

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14 September 2017

Ryan Baumann

Start: 2:00PM

PCR and Gel of 13 September 2017 minipreps

Purpose: To identify if Trg_PhoR and BBa_K1618037 Terminator are properly assembled in the UAP

Protocol:

PCR's of 9/13/17 MP1 (9/14/17 PCR1), MP2 (9/14/17 PCR2), and MP3 (9/14/17 PCR3), and an RFP positive control (9/14/17 PCR4) were run with the following conditions:

Component	Volume
Taq 2X Master Mix	10 μL
VR (10μM)	0.4 μL
VF2 (10μL)	0.4 μL
Samples	0.7 μL
MilliQ	8.5 μL
Total	20 μL

PCR’s were run using taq2kbp program following taq 2x master mix protocol. Annealing temperatures calculated using IDT’s annealing tempature protocoL All Colony PCR’s were run on a 1% agarose gel containing 2.0 μL ethidium bromide. Gel Layout:

Lane	Component
1	2 Log Purple DNA Ladder
3	9/14/17 PCR1
5	9/14/17 PCR2
7	9/14/17 PCR3
9	9/14/17 PCR4

Notes:

Stop: 6:00PM

Next:

Round two of golden gate assemblies.

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6 September 2017

Ryan Baumann

Start: 5:00PM

Golden Gate Assembly of BBa_K1618037 Terminator into UAP and Transformations

Purpose: To assemble plant terminator into the universale acceptor plasmid using BsmBI Type IIS endonuclease and transform on to chloramphenicol blue white screening plates. To transform amilCP chromoprotein (BBa_K592009) and eforRed Chromoprotein (BBa_K592012) on to chloramphenicol plates.

Protocol:

Reaction setup:

UAP	0.20 μL (~60 ng)
BBa_K1618037	5 μL (~50 ng)
10X T4 DNA Ligase Buffer	2 μL
T4 DNA Ligase	1 μL
BsmBI	1 μL
MilliQ Water	10.8 μL
	20 μL

Samples Ran: – 20 seconds @ 37°C, – (3 minutes @ 37°C, 4 minutes @ 16°C) X26 – 5 minutes @ 50°C – 5 minutes @ 80°C – 5 minutes @ 16°C 2 μL of the Terminator assembly, 1 μL of AmilCP, and 1 μL of eforRed were transformed using NEB® 5-alpha Competent E. coli (High Efficiency) protocol. The 1% and 10% dilutions were plated for each. AmilCP and eforRed were transformed on to chloramphenicol plates. The terminator assembly was plated on Chlor blue-white screening plates.

Stop: 10:30pm

Products:

Label	Source	Description
9/6/17 AmilCP	2017 Kit Plate 1 Well 19E	amilCP resuspended from kit plate
9/6/17 eforRed	2017 Kit Plate 7 Well 15I	eforRed resuspended from kit plate

Next:

Check for colony growth. Plasmid mini prep desired colonies.

---

2 September 2017

Ryan Baumann

Start: Colony PCR and Gel of 8/31/17 Transformants

Confirm colonies with properly assembled plant parts with promoter and terminator

Purpose: To find properly assembled plasmids of PhoB, PhoR, and TNTr3 with promoter and terminator within alpha 1 acceptor plasmid.

Protocol:

20uL Colony PCR's were run for 7 PhoB colonies, 7 TNTr3 colonies, and 6 PhoR colonies. Sample colony PCR

Component	Volume
Taq 2x Master Mix	10 μL
VR	0.8 μL
VF2	0.8 μL
MilliQ	8.4 μL
Colonies 1-20

PCR's were run using taq2kbp program following taq 2x master mix protocol with annealing temperatures calculated using IDT's annealing tempature protocol. All Colony PCR's were run on a 1% agarose gel containing 1.5 μL ethidium bromide. Gel Layout

Lane(s)	Sample
1	2 Log Purple Ladder
3-9	PhoB colonies 1-7
10-12	TNTr3 COlonies 1-3
13	2 Log Purple Ladder
15-18	TNTR3 4-7
19-24	PhoR colonies 1-6

Notes:

Stop:

Next:

Troubleshoot gel results.

---

1 September 2017

Benjamin Bleitz

Start: 1:00 pm

Preparation of Arabidopsis growth medium

Purpose: to prepare a proper growth medium for Arabidopsis in a laboratory setting

Protocol:

Component	Amount
Murashige and Skoog salts	2.15 g
1% Sucrose	5 ml
0.05% MES	0.25 ml
MilliQ H2O	total of 500 ml
KOH	Until pH adjusted to 5.7

Sucrose, MES and final solution all Autoclaved for 30 minutes on liquid cycle Plates poured

Stop: 3:30

Results:

18 plates prepared for plant growth

Products:

18 MS Mediums

Next:

Test growth mediums

---

31 August 2017

Ryan Baumann

Start: 3:00 PM

Addition of Promoter and Terminator to PhoR, PhoB, and TNTR3

Purpose: Addition of BBa_K1467101 Promoter and BBa_K1618037 Terminator to PhoR, PhoB, and TNTR3 into BBa_P10501 Alpha 1 acceptor plasmid via golden gate assembly using NEB golden gate assembly kit.

Protocol:

20 uL Reactions were set up following NEB golden gate assembly mix using 75ng of BBa_P10501 as the destination plasmid and 100ng of each insert. All samples were run at 37C for 1 hour followed by 55C for 5 minutes. 1 uL of each assembly were transformed using NEB® 5-alpha Competent E. coli (High Efficiency) protocol. 20% and 2% dilutions were then plated on Kanamysin selective blue white screening plates.

Stop: 7:00PM

Results:

Growth of white colonies was seen from assembly transformation.

Next:

Colony PCR from transformation plates to confirm proper assembly

---

23 August 2017

Ryan Baumann

Start: 3:00 PM

PCR of August 18th plasmid minipreps with VR and VF2 and gel extraction

Purpose: To verify correct assembly of fragments in the Universal Acceptor Plasmid and extract correct bands from gel

Protocol:

~100 ng of purified template DNA was amplified following the iGEM Lab Manual 2.5 PCR Protocol using Taq 2x MM, VR, VF2, and MilliQ H2O. A positive control was run using linearized Psb1T3 backbone. A negative control as run with a water template. Sample Reaction:

Sample	Volume
Taq 2X Master Mix	12.5 μL
VR	1 μL
VF2	1 uL
Plant Pho (100ng/1uL)	1 uL
Water	9.5 μL
Total	25 μL

Samples were run in thermocycler using Taq 2X master mix recommend protocol with appropriate annealing temperature for VR and VF2. All PCR's were run in a 1% agarose gel containing 2 μL ethidium bromide. Gel Layout:

Lane	Sample
1	2 Log Purple DNA ladder
3	Negative Control
4	TNTR3
5	Plant Pho
6	Positive Control
7	PhoB
8	PhoR

Bands containing Assembled TNTR3 (~1100BP), Plant Pho (~450BP), PhoB (~1100BP), and PhoR (~1800 BP) were cut from the gel and DNA was purified using Amicon Ultra-DA Kit for DNA extraction from agarose gel.

Notes:

Stop: 5:06 PM

Products:

Label	Source	Description
8/23/17 Plant Pho	8/18/17 plant Pho Miniprep	Extracted plant pho DNA from gel
8/23/17 Pho R	8/18/17 PhoR miniprep	extracted PhoR from gel
8/23/17 Pho B	8/18/17 PhoB miniprep	Extracted PhoB from Gel
8/23/17 TNTR3	8/18/17 tntr3 miniprep	Extracted TNTr3 from gel

Next:

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18 August 2017

Ryan Baumann

Start: 11:00am

Plasmid Miniprep of colonies from 8/9/17 COlony PCR's

Purpose: To isolate plasmids from colonies with correctly assembled PhoB, OhoR, Plant pho, and TNTR3

Protocol:

Colonies that showed positive bands from PCR were innoculated into broth cultures with chloramphenicol and grown up overnight. Plasmid DNA was purified using the MST iGEM Lab Manual 2.5 Kitless miniprep protocol and suspended in 40uL of TE buffer. 2uL of sample of was run on Thermoscientific Nanodrop 1000. Nanodrop results:

Sample	Concentration (ng/uL)	260nm/280nm
PhoB	263.2	1.84
PhoR	98.6	1.80
Plant Pho	99.8	1.86
TNTr3	236.0	1.82

Stop: 1:40PM

Products:

Label	Source	Description
8/18/17 MP1	PhoB Colonies from 6/26/17 Transformation	PhoB in the UAP
8/18/17 MP2	PhoR from 6/26/17 Transformation	PhoR in the UAP
8/18/17 MP3	Plant Pho from 6/26/17 Transformation	Plant Pho in the UAP
8/18/17 MP4	TNTR3 from 6/26/17 Transformation	TNTr3 in the UAP

Next:

PCR with VR and VF2 and extract band containing correctly assembled plasmid for sequencing. If correctly assembled, perform golden gate assembly to add Promoter and terminator to each plant part.

---

9 August 2017 to 10 August 2017

Ben Bleitz

Start: 2:00 PM of 9 August 2017

PCR and Gels of 18 Colonies from the PhoR Plate

Purpose: Correctly identify assembled of plasmid containing PhoR in universal acceptor

Protocol:

PCRs were conducted for marked colonies 1-18 on PhoR

Component	Volume
Taq 2x Master Mix	10μl
VR	0.8μl
VF2	0.8μl
MilliQ	8.4μl
Colonies 1-18

Samples were run through the Taq2bkp program and then frozen for the next day All Samples were run on a 1% agarose gel with .75 uL Ethidium Bromide with 2 Log Purple DNA ladder, the following day.

Stop: 1:00 PM of 10 August 2017

Results:

Next:

Inoculate culture from appropriate culture

---

8 August 2017

Ryan Baumann

Start: 4:00PM

Colony PCR of 6/26/17 TNTR3 transformation

Purpose: To identify correctly assembled plasmid containing TNTr3 in the universal acceptor plasmid 20 μL Sample reaction

Component	volume
Taq 2X Master Mix	10 μL
VR	0.8 μL
VF2	0.8 μL
MilliQ	8.4 μL
Colonies 1-16

A positive Control was conducted and run

Component	volume
Taq 2X Master Mix	10 μL
VR	0.8 μL
VF2	0.8 μL
MilliQ	7.4 μL
7/24/17 K608002	1 uL

A negative control PCR was conducted and run

Component	volume
Taq 2X Master Mix	10 μL
VR	0.8 μL
VF2	0.8 μL
MilliQ	8.4 μL
	20 μL

All Samples were run on a 1% agarose gel with .75 uL Ethidium Bromide wit 2 Log Purple DNA ladder.

Protocol:

PCR's of 16 colonies were run with VR and VF2 primers, Taq 2x Master mix, and MilliQ Water

Notes:

Stop: 9:00 PM

Results:

TBD

Next:

Examine Gel and find if colonies match correctly assembled plasmid

---

3 August 2017

Ben Bleitz

Start: 2:00

Running Gel of Plant Pho, PhoB, PhoR, TNTR3

Purpose: Running Gels of repeat PCR

Protocol:

Lane	Component
1	10 uL NEB 2 Log Purple DNA ladder
3	10 uL 7/27/17 PCR 5
5	10 uL 7/27/17 PCR 1
6	10 uL 7/27/17 PCR 2
7	10 uL 7/27/17 PCR 3
8
9	10 uL 7/24/17 K608002 (+ Control)
10	10 uL MilliQ Water (- Control)

Stop: 5:00

Results:

Somehow managed to repeat mistakes from last week. Will repeat, for a third and hopefully final time for last week.

Next:

DONT MESS UP

---

2 August 2017

Ben Bleitz

Start: 1:00 pm

PCR of 7/25 L1, 7/24 Plant Pho and 6/29 MP1 (PhoB), MP2 (PhoR), MP6 (TNTR3)

Purpose: Repeat of previous PCR (and eventual gel) from 27 June 2017. Gel scheduled to be completed following day (3 August 2017)

Protocol:

Plant Pho PCR:

Component	Volume
Q5 2x Master Mix	12.5 μL
VR	1μL
VF2	1 μL
7/24/17 Plant Pho	3 μL
MilliQ	7.5 μL
Total	25 μL

Pho B Pcr:

Component	Volume
Q5 2x Master Mix	12.5 μL
VR	1μL
VF2	1 μL
6/29/17 MP1	1.5 μL
MilliQ	9 μL
Total	25 μL

TNTR3 Pcr:

Component	Volume
Q5 2x Master Mix	12.5 μL
VR	1μL
VF2	1 μL
6/29/17 MP6	2.0 μL
MilliQ	8.5 μL
Total	25 μL

PhoR Pcr:

Component	Volume
Q5 2x Master Mix	12.5 μL
VR	1μL
VF2	1 μL
6/29/17 MP2	1.5 μL
MilliQ	9 μL
Total	25 μL

7/25/17 L1

Component	Volume
Q5 2x Master Mix	12.5 μL
VR	1μL
VF2	1 μL
7/25/17 L1	5 μL
MilliQ	5.5 μL
Total	25 μL

Notes:

PCR was completed. Gel will be ran following day

Stop: 4:20 pm (#Blazeit)

Results:

PCRs frozen after thermocycler completed. Will be determined upon gel electrophoresis completion on the following day.

Products:

Label	Source	Description
PCR 1	7/24/17 Plant Pho	7/24/17 Plant Pho amplified with VR and VF2
PCR 2	6/29/17 MPI	6/29/17 MPI amplified with VR and VF2
PCR3	6/29/17 MP6	6/29/17 amplified with VR and VF2
PCR4	6/29/17 MP2	6/29/17 amplified with VR and VF2
PCR5	7/25/17 L1	7/25/17 L1 amplified with VR and VF2

Next:

Run gel

---

27 June 2017

Erin Nischwitz, Ben Bleitz

Start: 11:00 am

PCR and gels of 7/25 L1, 7/24 Plant Pho and 6/29 MP1 (PhoB), MP2 (PhoR), MP6 (TNTR3)

Purpose: To verify assembly of these constructs

Protocol:

Five PCR's were ran with VR and VF2 using Q5 2x Master mix Protocol Plant Pho PCR:

Component	Volume
Q5 2x Master Mix	12.5 μL
VR	1μL
VF2	1 μL
7/24/17 Plant Pho	3 μL
MilliQ	7.5 μL
Total	25 μL

Pho B Pcr:

Component	Volume
Q5 2x Master Mix	12.5 μL
VR	1μL
VF2	1 μL
6/29/17 MP1	1.5 μL
MilliQ	9 μL
Total	25 μL

TNTR3 Pcr:

Component	Volume
Q5 2x Master Mix	12.5 μL
VR	1μL
VF2	1 μL
6/29/17 MP6	2.0 μL
MilliQ	8.5 μL
Total	25 μL

PhoR Pcr:

Component	Volume
Q5 2x Master Mix	12.5 μL
VR	1μL
VF2	1 μL
6/29/17 MP2	1.5 μL
MilliQ	9 μL
Total	25 μL

7/25/17 L1

Component	Volume
Q5 2x Master Mix	12.5 μL
VR	1μL
VF2	1 μL
7/25/17 L1	5 μL
MilliQ	5.5 μL
Total	25 μL

A 1% agarose gel was ran containing all five PCRs, a positive control, and a Negative control

Lane	Component
1	10 uL NEB 2 Log Purple DNA ladder
3	10 uL 7/27/17 PCR 5
5	10 uL 7/27/17 PCR 1
6	10 uL 7/27/17 PCR 2
7	10 uL 7/27/17 PCR 3
8	10 uL 7/27/17 PCR 4
9	10 uL 7/24/17 K608002 (+ Control)
10	10 uL MilliQ Water (- Control)

Notes:

-Accidentally used circular DNA as a positive control, so do not expect a valid + Control

Stop: 3:30

Results:

TBD

Products:

Label	Source	Description
PCR 1	7/24/17 Plant Pho	7/24/17 Plant Pho amplified with VR and VF2
PCR 2	6/29/17 MPI	6/29/17 MPI amplified with VR and VF2
PCR3	6/29/17 MP6	6/29/17 amplified with VR and VF2
PCR4	6/29/17 MP2	6/29/17 amplified with VR and VF2
PCR5	7/25/17 L1	7/25/17 L1 amplified with VR and VF2

Next:

Trouble Shoot

---

24 July 2017

Ryan Baumann

Start: 11:00 AM

Plasmid Miniprep of 7/21/17 Transformations

Purpose: Prepare plasmids for future use

Protocol:

Kitless miniprep protocol from igem lab manual version 2.5 Plasmids were suspended in 35 μl of 1x TE buffer

Stop: 12:40PM

Results:

Samples tested on Thermoscientific Nanodrop 1000

Label	DNA Conc.	260/280
BBa_B0015	893.5 ng/uL	1.95
BBa_K608002	573.5 ng/uL	1.88
Plant Pho	165.7 ng/uL	1.93

Skype Backpack

Products:

Label	Source	Description
7/24/17 B0015	2017 Kit Plate 3 well 3F	B0015 Terminator in psb1c3
7/24/17 Plant Pho	Plant Pho top and bottom strand	Plant pho in UA plasmid
7/24/17 K608002	2017 Kit Plate 1 well 3O	K608002 Promoter and RBS in psb1c3

Next:

---

25/June/2017

Ben Bleitz

Start: 1:30

Digestion and Ligation of BBa_K608002 and TNTR3_E.Coli

Purpose: To prepare DNA Samples for Transformations

Protocol:

Two double digestions were performed BBa_K608002 Double digestion

Component	Volume
ECORI Enzyme	1 μl
SpeI Enzyme	1 μl
K608002 Insert	3.5 μl
Tango Buffer	2.5 μl
MilliQ H2O	17 μl

TNT_E.Coli Double Digestion

Component	Volume
ECORI Enzyme	1 μl
Xbal Enzyme	1 μl
TNTR3_E.Coli Vector	5 μl
Tango Buffer	2.5 μl
MilliQ H2O	15.5 μl

Products were 25/7/2017 d1 and 25/7/2017 d2 Ligation was then performed

Component	Volume
25/7/2017 d1 (insert)	1.5 μl
25/7/2017 d2 (vector)	2 μl
T4 Ligase Buffer	2 μl
T4 Ligase	1 μl
13.5 MilliQ H2O	13.5

Stop: 5:30

Products:

Label	Source	Description
25/7/2017 d1	BBa_K608002	K608002 cut with ECORI & SPeI
25/7/2017 d2	TNTR3_E.Coli	TNTR3_E.Coli cut with ECORI & Xbal
25/7/2017 L1	d1 and d2	DNA formed from d1 insert and d2 vector

Next:

PCR with VR and VF 2 to confirm products

---

20/7/2017

Ben Bleitz

Start: 2:30

Bacterial promoter/ terminator and plant pho Transformations

Purpose: Transform components for later prep and kit plate

Protocol:

Transformations were performed using NEB® 5-alpha Competent E. coli (High Efficiency) protocol.

Transformant	DNA Volume	Plate Dilutions
Plant Pho	1μl	2 and 20%
BBa_B0015	1μl	2 and 20%
BBa_K608002	1μl	2 and 20%

Stop: 5:30

Results:

All samples in incubation. Results will be noted at the end of 24 hour incubation period

Products:

Label	Source	Description
BBa_B0015	2017 Kit Plate 3 well 3F	Transformed on chlorophenicol plate
BBa_K608002	2017 Kit Plate 1 well 3O	Transformed on chlorophenicol plate
Plant Pho	Plant Pho top and bottom strand	Transformed on Blue/White chlorophenicol plate

Next:

Inoculate in broth culture with appropriate antibiotic for future miniprep.

---

10 July 2017

Ryan Baumann

Start: 12:30

Golden Gate assembly of Plant Pho Top and Bottom strand into UA plasmid

Purpose: To assemble PlantPho Oligos into UAP to prepare for assembly of Transcriptional Unit

Protocol:

UA MP1 (100ng)	0.3 μL
PlantPho Top Strand (100 ng)	1 μL
PlantPho Bottom Strand (100 ng)	1 μL
10X T4 DNA Ligase Buffer	2 μL
T4 DNA Ligase	1 μL
BsmBI	1 μL
MilliQ H2O	13.7 μL
	20 μL

Ran at: – 20 seconds @ 37°C, – (3 minutes @ 37°C, 4 minutes @ 16°C) X26 – 5 minutes @ 50°C – 5 minutes @ 80°C – 5 minutes @ 16°C

Stop: 5:00

Products:

Label	Source	Description
7/10/17	Plant Pho Top & Bottom Strand Oligos	Plant Pho Oligos cut with BsmBI and Ligated into UAP

Next:

Transform on to Chlor Blue white Screening Plates

---

29 June 2017

Ryan Baumann, Ben Bleitz

Start: 1:30

Miniprep of 6/26/17 transformations

Purpose: Prepare plasmids for future use

Protocol:

Kitless miniprep protocol from igem lab manual version 2.5 Plasmids were suspended in 35 μl of 1x TE buffer

Stop: 3:30

Results:

Samples tested on Thermoscientific Nanodrop 1000

Label	DNA Concentration (ng/μl)	260/280
MP1	412.7	1.84
MP2	399.6	1.83
MP3	290.2	1.82
MP4	722.4	1.80
MP5	406.4	1.81
MP6	307.4	1.84

Products:

Label	Source	Description
6/29/17 MP1	PhoB-VP64 Transformation plate	PhoB-VP64 transformed into UA Plasmid
6/29/17 MP2	Trg-PhoR Transformation plate	Trg-PhoR transformed into UA Plasmid
6/29/17 MP3	Golden Braid Omega 1 Plasmid Transformation plate	Golden Braid Omega 1 plasmid
6/29/17 MP4	Golden Braid Omega 2 Plasmid Transformation plate	Golden Braid Omega 2 plasmid
6/29/17 MP5	TNTR3 E. Coli Transformation plate	TNTR3 E. Coli in PSB1C3 backbone
6/29/17 MP6	TNTR3 Transformation plate	TNTR3 transformed into UA plasmid

Next:

TBD

---

6/26/2017

Ryan Baumann, Ben Bleitz, Erin Nischwitz

Start: 5:00 pm

Chemical Transformations

Purpose: To transform PhoB-VP64, Trg_PhoR, Plant Pho, and TNTR3 on to chloramphenicol blue white screening plates. To transform TNTR3_E Coli (6/22/17 L1) on to chloramphenicol plate To transform Golden Braid Omega 1 and Omega =1 plasmids on to Streptomycin plates.

Protocol:

Transformations were performed using NEB® 5-alpha Competent E. coli (High Efficiency) protocol.

Transformant	DNA volume	Plate Dillutions
TNTR3	1 μL	2% and 20%
Trg_PhoR	1 μL	2% and 20%
PhoB_VP64	1 μL	2% and 20%
Plant Pho	2 μL	2% and 20%
6/22/17 L1 (TNTR3 E coli)	3 μL	5% and 50%
Omega 1	1 μL	2% and 20%
Omega Two	1 μL	2% and 20%

Stop: 7:45

Next:

Check for colonies and inoculate cultures

---

6/26/2017

Golden Gate Assembly into Universal Acceptor Plasmid

Start: 12:00pm

Assembly of PhoB_VP64, Trg_PhoR, TNTR3, and Plant Pho into BBa_P10500 plasmid.

Purpose: To assemble gblocks into UAP to prepare for assembly of Transcriptional Unit

Protocol:

Trg_PhoR-Gblock:

UA MP1 (100ng)	0.30 μL
Trg_PhoR (100ng)	1 μL
10X T4 DNA Ligase Buffer	2 μL
T4 DNA Ligase	1 μL
BsmBI	1 μL
MilliQ H2O	14.7 μL
	20 μL

PhoB_VP64-gblock

UA MP1 (100ng)	0.30 μL
PhoB_VP64 (100ng)	1 μL
10X T4 DNA Ligase Buffer	2 μL
T4 DNA Ligase	1 μL
BsmBI	1 μL
MilliQ H2O	14.7 μL
	20 μL

TNT_R3-gblock

UA MP1 (100ng)	0.30 μL
TNT_R3 (100ng)	1 μL
10X T4 DNA Ligase Buffer	2 μL
T4 DNA Ligase	1 μL
BsmBI	1 μL
MilliQ H2O	14.7 μL
	20 μL

PlantPho Oligos Top and Bottom Strands

UA MP1 (100ng)	0.30 μL
PlantPho Top Strand (100 ug)	1μL
PlantPho Bottom Strand (100 ug)	1μL
10X T4 DNA Ligase Buffer	2 μL
T4 DNA Ligase	1μL
BsmBI	1μL
MilliQ H2O	13.7 μL
	20 μL

All Samples Ran: - 20 seconds @ 37°C, - (3 minutes @ 37°C, 4 minutes @ 16°C) X26 - 5 minutes @ 50°C - 5 minutes @ 80°C - 5 minutes @ 16°C

Notes:

100 μg of top and bottom strand oligos were accidentally used instead of 100ng.

Stop: 4:45 PM

Products:

Label	Source	Description
6/26 TNTR3	TNTR3-GBlock	TNTR3-GBlock cut with BsmBI and Ligated into UAP
6/26 Plant Pho	Plant Pho Top & Bottom Strand Oligos	Plant Pho Oligos cut with BsmBI and Ligated into UAP
6/26 PhoR	Trg_PhoR-Gblock	Trg_PhoR-Gblock cut with BsmBI and Ligated into UAP
6/26 PhoB	PhoB_VP64 Gblock	PhoB_VP64 Gblock cut with BsmBI and Ligated into UAP

Next:

Transform assembled plasmids on to Blue White Screening Plates with appropriate antibiotic resistance

---

6/26/2017

Golden Gate Assembly into Universal Acceptor Plasmid

Start: 12:00pm

Assembly of PhoB_VP64, Trg_PhoR, TNTR3, and Plant Pho into BBa_P10500 plasmid.

Purpose: To assemble gblocks into UAP to prepare for assembly of Transcriptional Unit

Protocol:

Trg_PhoR-Gblock:

UA MP1 (100ng)	0.30 μL
Trg_PhoR (100ng)	1 μL
10X T4 DNA Ligase Buffer	2 μL
T4 DNA Ligase	1 μL
BsmBI	1 μL
MilliQ H2O	14.7 μL
	20 μL

PhoB_VP64-gblock

UA MP1 (100ng)	0.30 μL
PhoB_VP64 (100ng)	1 μL
10X T4 DNA Ligase Buffer	2 μL
T4 DNA Ligase	1 μL
BsmBI	1 μL
MilliQ H2O	14.7 μL
	20 μL

TNT_R3-gblock

UA MP1 (100ng)	0.30 μL
TNT_R3 (100ng)	1 μL
10X T4 DNA Ligase Buffer	2 μL
T4 DNA Ligase	1 μL
BsmBI	1 μL
MilliQ H2O	14.7 μL
	20 μL

PlantPho Oligos Top and Bottom Strands

UA MP1 (100ng)	0.30 μL
PlantPho Top Strand (100ng)	1μL
PlantPho Bottom Strand (100ng)	1μL
10X T4 DNA Ligase Buffer	2 μL
T4 DNA Ligase	1μL
BsmBI	1μL
MilliQ H2O	13.7 μL
	20 μL

All Samples Ran: - 20 seconds @ 37°C, - (3 minutes @ 37°C, 4 minutes @ 16°C) X26 - 5 minutes @ 50°C - 5 minutes @ 80°C - 5 minutes @ 16°C

Stop: 4:45 PM

Products:

Label	Source	Description
6/26 TNTR3	TNTR3-GBlock	TNTR3-GBlock cut with BsmBI and Ligated into UAP
6/26 Plant Pho	Plant Pho Top & Bottom Strand Oligos	Plant Pho Oligos cut with BsmBI and Ligated into UAP
6/26 PhoR	Trg_PhoR-Gblock	Trg_PhoR-Gblock cut with BsmBI and Ligated into UAP
6/26 PhoB	PhoB_VP64 Gblock	PhoB_VP64 Gblock cut with BsmBI and Ligated into UAP

Next:

Transform assembled plasmids on to Blue White Screening Plates with appropriate antibiotic resistance

---

23/6/17

Ben Bleitz

Start: 2:50pm

Ligation of PsbIC3 (Vector) and 6/21/17 DI (insert)

Purpose: To create recombinant DNA for transformation

Protocol:

T4 Ligase Buffer	2μL
PsbIC3 (Vector)	4μL
6/21/17 DI (Insert)	3μL
MilliQ H2O	10μL
T4 DNA Ligase	1μL

Thermal Cycler 16°C for 30 minutes 80°C for 20 minutes

Stop: 4:00pm

Results:

6/23/17 L1

Next:

E. Coli Transformation

---

6/21/2017

Ben Bleitz

Start: 2:00

Double Digestion of TNT R3_E.Coli

Purpose: To prepare TNT R3_E.Coli for submission into the registry

Protocol:

Volume	Component
2.5 μL	10X Tango Buffer
1 μL	ECORI
1 μL	PSTI
15 μL	TNTR3_E.Coli (10 ng/μL)
5.5 μL	MilliQ H2O
25 μL	in Total

Ran @ 37° C for 1 hour Heat kill @ 80°C for 30 minutes

Stop: 4:00 PM

Products:

Label	Source	Description
6/21/17 D1	TNTR3_E.Coli	TNTR3_E.Coli cut with ECORI and PSTI

Next:

Ligate into PsbIC3