Team:Lethbridge/Notebook/Lab Work/August

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Feel free to look around our notebook!

Here you can check out the work we have done in the lab, click on a month to take a look!

Contents

1 August

1.1 August 3, 2010

1.2 August 3, 2010 Evening

1.3 August 4, 2010

1.4 August 6, 2010

1.5 August 6, 2010 Evening

1.6 Aug 9, 2010

1.7 Aug 9,2010 Evening

1.8 Aug 10, 2010

1.9 Aug 10, 2010 Evening

1.10 Aug 11, 2010

1.11 Aug 12, 2010

1.12 Aug 13, 2010

1.13 Aug 14, 2010

1.14 Aug 14, 2010 Evening

1.15 Aug 15, 2010

1.16 Aug 15, 2010 Evening

1.17 Aug 16, 2010

1.18 Aug 16, 2010 Evening

1.19 Aug 17, 2010

1.20 Aug 17, 2010 Evening

1.21 Aug 19, 2010

1.22 Aug 20, 2010

1.23 Aug 23, 2010

August

August 3, 2010

(in Lab: HB, AV, JV)

Objective: To restrict pBAD, sRBS, mRBS, TetR, dT and pTet for the assembly of pBAD-sRBS-TetR-dT-pTet

Method: Used Restriction of Plasmid DNA protocol.

A front verctor was made made in sRBS ,mRBS, dT plasmids using EcoRI and Xbal enzymes
pDNA that was cut out of plasmid for a front vector was pBAD, TetR, dT and pLacI using EcoRI and SpeI enzymes
A back vector was made in sRBS and mRBS plasmids with PstI and SpeI
pDNA that was cut out of plasmid for a back vector was TeTR and it was restricted with XbaI and PstI

Construct pDNA buffer Enzymes

pBAD-sRBS/mRBS pBAD Red EcoRI and SpeI

pBAD-sRBS/mRBS sRBS Orange XbaI and EcoRI

pBAD-sRBS/mRBS mRBS Orange XbaI and EcoRII

sRBS/mRBS-TetR sRBS Red PstI and SpeI

sRBS/mRBS-TetR mRBS Red PstI and SpeI

sRBS/mRBS-TetR TetR Tango XbaI and PstI

TetR-dT TetR Red EcoRI and SpeI

TetR-dT dT Orange XbaI and EcoRI

dT-pTet dT Red EcoRI and SpeI

dT-pTet pTet Orange XbaI and EcoRI

pLAcI-sRBS/mRBS pLacI Red EcoRI and SpeI

pLAcI-sRBS/mRBS sRBS Orange XbaI and EcoRI

pLAcI-sRBS/mRBS mRBS Orange XbaI and EcoRI

Mms6-PET28(a) PET28(a) Orange NotI

For all reactions
158 (µL) Milli-Q H₂O
10 (µL) Buffer
0.5(µL) of each enzyme
10 (µL) pDNA

Restriction was incubated for 1 hour at 37^oC

Objective: Run PCR of pBAD, TetR, dT, pLacI, and mms6.
Method: Used PCR thermocycler iGEM program 7

Component Volume per tube (µL) Master Mix (x6)

MilliQ H₂O 41.8 250.8

10x Pfu Buffer with MgSO₄ 5 30

dNTPs 1 6

Forward Primer 0.5 3

Reverse Primer 0.5 3

Template DNA 1 -

Pfu DNA polymerase 0.2 -

Total 50 292.8

Added 48.8 µL of Master Mix to each PCR reaction

Objective: Complete maxipreps of Lumazine (K249002), EYFP (E0030), and ECFP (E0020) and run on 1% agarose gel.
Method:

Lane Sample Components (µL)

1 1 kb ladder 2 loading dye (5x) + 0.5 ladder + 3.5 MilliQ H₂O

2 ECFP 2 DNA + 2 loading dye (5x) + 2 MilliQ H₂O

3 EYFP 2 DNA + 2 loading dye (5x) + 2 MilliQ H₂O

4 Lumazine 2 DNA + 2 loading dye (5x) + 2 MilliQ H₂O

Ran at 100V for 42 minutes. Stained in EtBr for 15 minutes.

Results: AGAROSE GEL PICTURE

Objective: Ligate dT into pSB1C3.
Method:

PCR amplify and purify both pSB1C3 and dT
Restrict both with EcoRI and PstI
Restrict pSB1C3 with DpnI
Ligate pSB1C3 and dT

Restriction:

Restriction MilliQ H₂O (µL) Buffer Orange (µL) pDNA (µL) Enzyme (µL)

pSB1C3 79.25 10 10 0.25 EcoRI + 0.25 PstI + 0.25 DpnI

pSB1C3 control 80 10 10 -

dT 79.50 10 10 0.25 EcoRI + 0.25 PstI

dT control 80 10 10 -

Restriction were incubated at 37^oC for 90 minutes.
Enzymes were heat killed for 20 minutes at 80^oC.

August 3, 2010 Evening

(in lab: KG, JS)

Objective: To run 1.5% agarose of restrictions: pBAD, sRBS, mRBS, TetR, dT and pTet for the assembly of pBAD-sRBS-TetR-dT-pTet

Method: Used a 1.5% agarose gel with 2 (µL) of loading dye and 10 (µL) of pDNA.

Lane Contents Result
1 1kb ladder
2 pTet (XbaI, EcoRI) good
3 pTet (orange buffer) ---
4 dT (XbaI, EcoR) no good
5 dT (orange buffer) ---
6 mRBS (SpeI, PstI) good
7 mRBS (red buffer) ---
8 sRBS (SpeI, PstI) good
9 sRBS (red buffer) ---
10 mRBS (XbaI, EcoRI) good
11 mRBS (orange buffer) ---
12 sRBS (XbaI, EcoRl) good
13 sRBS (orange buffer) ---
14 pet28(a) good
15 100 bp ladder
16 PSB1C3 not good
17 PSB1C3 restriction digest ---

Lane Contents Result
1 TetR (EcorI, SpeI) good
2 TetR (red buffer) ---
3 pLacI (EcoRI, SpeI) good
4 pLacI (red buffer) ---
5 Mms6 can not tell
6 Mms6 control ---
7 TetR (Xbal, PstI) good
8 TetR (tango buffer) ---
9 pBAD (EcoRI, SpeI) good
10 pBAD (red buffer) ---
11 dT (EcoRI, SpeI) good
12 dT (red buffer) ---
13 pLacI (2) ?
14 dT control not good
15 dT restriction
16 100 bp ladder
17 dT PCR product good
18 Mms6 PCR product good
19 pBAD PCR product good
20 pLacI PCR product good

Objective: To ligate: pBAD-sRBS/mRBS, sRBS/mRBS-TetR, TetR-dT, dT-pTet, pLacI-sRBS/mRBS, Mms6-ptet28(a), dT-PSB1C3

Method: Ligation of Plasmid DNA

15 (µL) pDNA in plasmid, and 15 (µL) of pDNA biobrick)

August 4, 2010

(in lab: JV)

Objective: PCR analysis of ligation product of August 3, 2010

Ligations
pBAD-mRBS
pBAD-SRBS
SRBS-tetR
mRBS-TetR
dt-pTet
mms6-pET-28a
dt-pSBIC3
pLacI-SRBS

Controls
pBAD
TetR
TetR
pLacI
mms6

Method:
PCR: Thermocycler set to iGEM program 7

Component 1X(µL) Master Mix(x16)(µL)
Milli-Q H₂O 41.8 668.6
10x Pfu Buffer with MgSO₄ 5 80
dNTPs 1 16
Forward Primer 0.5 8
Reverse Primers 0.5 8
Template DNA 1 16
Pfu polymerase 0.2 3.2

2.5% agarose gel(1x TAE)

lane contents Successful Ligation ?
1 50bp ladder ---
2 dt pSBIC3 ---
3 dt pTet x
4 dt control ---
5 sRBS-TetR x
6 mRBS-TetR ?
7 TetR control ---
8 pLacI-mRBS x
9 pLacI-sRBS ?
10 pLacI control ---
11 Mms6 pET-28a no band
12 Mms6 control ---
13 pBad-SRBS x
14 pBad-mRBS x
15 pBad control ---

Ran at 100V for 70 minutes.

Results: AGAROSE GEL PICTURE

Objective: Transform the successful ligations

Method: used Competent Cell Transformation protocol

changes:
used 50µL aliquottes of DH5&alpha
did not pipette up and down once, the cells were just swirled 3 times
added 400µL SOC media, shoock at 37⁰C for 90 min
platted 250µL and 150µL

Incubated from 12:00AM to 4;00 PM

results
contents &250µL 150µL
dt-pTet good x
- control x x
mms6-pET-28a good good
dt-pSBIC3 x x
mRBS-TetR good good
pLacI-mRBS good good
SRBS-TetR x x
pBAD-SRBS good good
+ contol good good

August 6, 2010

In Lab: JV
Objective: Put lumazine synthase gene and mms6 into pET-28(A) for future overexpression.

Method: Restrict mms6 from Mr. Gene with NotI. Large quantities of DNA can be used so a gel extraction of the part can be done. PCR lumazine synthase out of its backbone. Restrict off the extra DNA fragments from the PCR with NotI. Restrict pET-28A with notI. Ligate.

Restriction:

Restriction MilliQ H₂O (µL) Buffer Orange (µL) pDNA (µL) Enzyme (µL)

mms6 799.5 100 100 1 NotI

PCR:

Component 1X(µL)
Milli-Q H₂O 41.85
10x Pfu Buffer with MgSO₄ 5
dNTPs 1
Forward Primer (VF2) 0.5
Reverse Primers (VR) 0.5
Template DNA (Lumazine Synthase) 1
Pfu polymerase 0.2

2% Agarose Gel for Gel Extraction of mms6:

lane sample loaded
1 mms6 restricted with NotI 1 mL sample
2 mms6 unrestricted control 10(µL) sample, 2(µL)dye
3 50bp ladder 2(µL) ladder, 2(µL) dye, 8(µL) H₂0
3 1 kb ladder 2(µL) ladder, 2(µL) dye, 8(µL) H₂0

Gel ran for 60 minutes at 100 V. Small & faint band was slightly visible. Gel extraction was carried out using QIAGEN method. Eluted to 12 (µL).

August 6, 2010 Evening

Objective: Attempt colony pcr for rapid screening

Method: Followed two protocols from openwet

Knight Protocol

place 20(µL) sterile H₂O in 0.6mL sterile tube
with P10 pipette set to 3(µL) dip tip into colony
place pipette tip into water and pipette up and down 20 times(this can be stored at 4⁰C for inoculation of overnight 5mL cultures)

Endy Protocol
place 50(µL) sterile H₂O in 0.6mL sterile tube
with PLO pipette (set 3(µL)) dip sterile tip into colony
place pipette tip into water and pipette up and down 20 times

Knight cont'd Endy cont'd
setup 20(µL) reaction setup 20(µL) reaction
1(µL) colony suspension 2(µL) colony suspension
2(µL) 10x p.fu (+Mg SO₄ 2(µL) 10x p.fu (+Mg SO₄
2(µL) dNTP 2(µL) dNTP
1.25(µL)VF2 Primer (10(µM) 0.75(µL)VF2 Primer (10(µM)
1.25(µL)VF Primer (10(µM) 0.75(µL)VF Primer (10(µM)
0.2(µL) Pfu polymerase 0.2(µL) Pfu polymerase
11.8 Milli-Q H₂O 12.6 Milli-Q H₂O

as control for each rxn used equal volume of mRBS maxiprep

Knight cont'd Endy cont'd
cycling conditions cycling conditions
95⁰C for 15 minutes 95⁰C for 6 minutes
*94⁰C for 30 seconds **95⁰C for 30 seconds
*56⁰C for 30 seconds **56⁰C for 30 seconds
*68⁰C for 1 minutes **70⁰C for 1 minutes
68⁰C for 20 minutes 70⁰C for 10 minutes

(*) were run 39 times
(**) were run 35 times

Made the following program (called COLONYY) Lid preheat 98⁰C

98⁰C for 15 minutes
98⁰C for 30 seconds
56⁰C for 30 seconds
68-70⁰C gradient for 1 minute
68-70⁰C gradient for 20 minute
4⁰C indefinte

bold selections were cycled 39 times

Objective: Analyzed PCR products on 2.5% TAE Agarose gel.

lane sample loaded
1 50bp ladder 1(µL) ladder, 1(µL) dye, 4(µL) H₂0
2 Knight control 5(µL) sample, 1(µL)dye
3 Knight colony 5(µL) sample, 1(µL)dye
4 Endy control 5(µL) sample, 1(µL)dye
5 Endy colony 5(µL) sample, 1(µL)dye
6 50bp ladder 1(µL) ladder, 1(µL) dye, 4(µL) H₂0
7 lumazine(justin's) 5(µL) sample, 1(µL)dye

Repeat gel with template controls

lane sample loaded
1 50bp ladder 0.5(µL) ladder, 2(µL) dye, 9.5(µL) H₂0
2 Endy Template 5(µL) colony suspension, 1(µL)dye, 4(µL) H₂0
3 Endy mRBS Control (PLR) 5(µL) sample, 1(µL)dye
4 Endy mRBS-TetR colony(PCR) 5(µL) sample, 1(µL)dye
4 mRBS template 0.5(µL) sample, 1(µL)dye, 4(µL) H₂0
5 Knight Template 0.25(µL) colony suspension, 1(µL)dye, 4(µL) H₂0
6 Knight mRBS control 5(µL) ladder, 1(µL) dye
7 Knight-mRBS-TetR colony 5(µL) sample, 1(µL)dye
1 1Kb ladder 0.5(µL) ladder, 2(µL) dye, 9.5(µL) H₂0

ran at 100V for 75 minutes

Aug 9, 2010

(In Lab: HB)
Objective: Run PCR of mRBS-xylE I1 and I2 and lumazine.

Method:
PCR: Thermocycler set to iGEM program 7

Component 1X(µL) Master Mix(x4)(µL)
Milli-Q H₂O 41.8 167.2
10x Pfu Buffer with MgSO₄ 5 20
dNTPs 1 4
Forward Primer (VF2) 0.5 2
Reverse Primers (VR) 0.5 2
Template DNA 1
Pfu polymerase 0.2

Added 48.8µL Master Mix to each reaction tube.
Objective: Run 2% agarose gel to confirm PCR of mRBS-xylE I1 and I2 and lumazine worked.

lane sample loaded
1 50bp ladder 0.5(µL) ladder, 1(µL) dye, 6.5(µL) H₂0
2 mRBS-xylE I1 5(µL) sample, 2(µL)dye
3 mRBS-xylE I2 5(µL) sample, 2(µL)dye
4 Lumazine 5(µL) sample, 2(µL)dye

Ran at 100 V for 45 minutes. mRBS-xylE did not amplify while lumazine did amplify.
GEL PICTURE!

(In Lab: AV)

Objective: Prepared glycerol stocks & Miniprepped the following using the Qiagen Protocol.

pLacI-mRBS Colony 1
pLacI-mRBS Colony 2
pLacI-sRBS Colony 2
pLacI-sRBS Colony 3
pBAD-mRBS Colony 1
pBAD-mRBS Colony 2
pBAD-sRBS Colony 1
pBAD-sRBS Colony 2
dT-pTet Colony 1
dT-pTet Colony 3
mRBS-TetR Colony 1
mRBS-TetR Colony 3

Objective: To determine which of the previous ligations worked.

Method: Restricted with single cutter and double cutter.

Restriction Reaction (SINGLE)

Ingredient Volume(µL)

MilliQ H₂0 Water 15.75

Orange Buffer (10x) 2

pDNA 2

EcoRI 0.25

Restriction Reaction (DOUBLE)

Ingredient Volume(µL)

MilliQ H₂0 Water 15.50

Orange Buffer (10x) 2

pDNA 2

EcoRI 0.25

PstI 0.25

Unrestricted Control

Ingredient Volume(µL)

MilliQ H₂0 Water 16

Orange Buffer (10x) 2

pDNA 2

DNA was restricted for 1 hour at 37^oC.

Analyzed results on a 2% agarose gel with 2 (µL) of loading dye and 10 (µL) of pDNA. Load order as follows:

Lane Contents
1 1kb ladder
2 50 bp ladder
3 dT-pTet 1 DRD
4 dT-pTet 1 SRD
5 dT-pTet 1 URD
6 dT-pTet 3 DRD
7 dT-pTet 3 SRD
8 dT-pTet 3 URD
9 pLacI-mRBS 1 DRD
10 pLacI-mRBS 1 SRD
11 pLacI-mRBS 1 URD
12 pLacI-mRBS 2 DRD
13 pLacI-mRBS 2 SRD
14 pLacI-mRBS 2 URD
15 pLacI-sRBS 1 DRD
16 pLacI-sRBS 1 SRD
17 pLacI-sRBS 1 URD
18 pLacI-sRBS 3 DRD
19 pLacI-sRBS 3 SRD
20 pLacI-sRBS 3 URD

Lane Contents
1 1 kb ladder
2 50 bp ladder
3 pBAD-mRBS 1 DRD
4 pBAD-mRBS 1 SRD
5 pBAD-mRBS 1 URD
6 pBAD-mRBS 2 DRD
7 pBAD-mRBS 2 SRD
8 pBAD-mRBS 2 URD
9 pBAD-sRBS 1 DRD
10 pBAD-sRBS 1 SRD
11 pBAD-sRBS 1 URD
12 pBAD-sRBS 2DRD
13 pBAD-sRBS 2 SRD
14 pBAD-sRBS 2 URD
15 mRBS-TetR 1 DRD
16 mRBS-TetR 1 SRD
17 mRBS-TetR 1 URD
18 mRBS-TetR 3 DRD
19 mRBS-TetR 3 SRD
20 mRBS-TetR 3 URD

GEL PICTURE!

Aug 9,2010 Evening

(In Lab: JV, AS)

Objective: To ligate: lumazine into vector upstream of dT. Lumazine and mms6 into pET28a.

Method:

Restrictions

Restrict Lumazine wit EcoRI and SpeI (Red Buffer)

Restrict the dT with XbaI and EcoRI (Orange Buffer)

Restrict Lumazine Synthase with NotI (Red Buffer)

Set up reactions as follows:

Component Volume (µL)

MilliQ H₂O 15.6 or 15.8

Buffer 2

pDNA 2

Enzyme 0.20 + 0.20

Incubated reactions for 60 minutes at 37^oC

Ligation
Reaction set up as follows:
T4 DNA ligase - 0.25µL

DNA 1 - 8µL

DNA 2 - 8µL

10x Ligation Buffer - 2µL

MilliQ H₂O - 1.75µL

Incubated reactions overnight at room temperature.
Aug 10, 2010

(In Lab: JV)

Objective: Reran large gel from Aug 9/2010.

Load order was as follows:

Lane Contents
1 50 bp ladder
2 pBAD-mRBS 2 URD
3 pBAD-mRBS 2 SRD
4 pBAD-mRBS 2 DRD
5 pLacI-sRBS 3 URD
6 pLacI-sRBS 3 SRD
7 pLacI-sRBS 3 DRD
8 pLacI-mRBS 2 URD
9 pLacI-mRBS 2 SRD
10 pLacI-mRBS 1 DRD
11 dT-pTet 3 URD
12 dT-pTet 3 SRD
13 dT-pTet 3 DRD
14 pBAD-mRBS 1 URD
15 pBAD-mRBS 1 SRD
16 pBAD-mRBS 1 DRD
17 pLacI-sRBS 2 URD
18 pLacI-sRBS 2 SRD
19 pLacI-sRBS 2 DRD
20 1 kb ladder

Lane Contents
1 50 bp ladder
2 pLacI-mRBS 1 URD
3 pLacI-mRBS 1 SRD
4 pLacI-mRBS 1 DRD
5 dT-pTet 1 URD
6 dT-pTet 1 SRD
7 dT-pTet 1 DRD
8 mRBS-TetR 3 URD
9 mRBS-TetR 3 SRD
10 mRBS-TetR 3 DRD
11 mRBS-TetR 1 URD
12 mRBS-TetR 1 SRD
13 mRBS-TetR 1 DRD
14 pBAD-sRBS 2 URD
15 pBAD-sRBS 2 SRD
16 pBAD-sRBS 2 DRD
17 pBAD-sRBS 1 URD
18 pBAD-sRBS 1 SRD
19 pBAD-sRBS 1 DRD
20 1 kb ladder

GEL PICTURE!

(In Lab: JV)

Objective: Determine which ligations/transformations worked from 08/04/10.

Method: Colony PCR.

A: dT-pTet (1-10) B: pBAD-sRBS (1-10) C: pLacI-sRBS (1-10) D: pBAD-mRBS (1-10) E: mRBS-TetR (1-10) F: pLacI-mRBS (1-10)
Pick colony with pipette set at 3(µL) Pipette colony up and dow in 20(µL) sterile Milli-Q water. Will use 96 well plate.
PCR- Conditions: 1. 95^oC for 15 min 2. 98^oC for 20 sec 3. 55^oC for 40 sec 4. 72^oC for 2 min 5. 72^oC for 20 min 6. 4^oC infinitely
Reaction Mixture -
Method:
PCR: Thermocycler set to iGEM program 7

Component 1X(µL) Master Mix(x65)(µL)
Milli-Q H₂O 10.9 708.5
5X Phusion HF Buffer 4 260
dNTPs 1 65
Forward Primer (VF2) 1 65
Reverse Primers (VR) 1 65
Colony Template 2
Phusion polymerase 0.17 11.05

Controls: sRBS, pTET & mRBS - will PCR these to compare size using same conditions as above.
GEL PICTURE!

Aug 10, 2010 Evening

(In Lab: ADS)

Objective: PCR amplify xylE from mRBS-xylE for creation of xylE BioBrick

Method: 20µL reactions

PCR- Conditions: 1. Initial Denaturation 98^oC for 30 sec 2. Denaturation 98^oC for 10 sec 3. Anneal (51^oC, 55^oC,59.8^oC, 64.6^oC, 69.1^oC, 71^oC) for 30 sec 4. Extend 72^oC for 30 sec 5. Final Extend 72^oC for 10 min 6. Held 4^oC for 30 hours
6 tubes in gradient PCR.

Component 1X(µL) Master Mix(x6.5)(µL)
Milli-Q H₂O 8.8 57.2
5X Phusion HF Buffer 4 26
dNTPs 2 13
Forward Primer 2 13
Reverse Primer 2 13
Template DNA 1 6.5
Polymerase 0.2 1.3

Ran samples on 1.5% agarose gel (1X TAE) for 60 minutes at 100V.
GEL PICTURE!

Aug 11, 2010

(In Lab: AS, JV, TF)

Objective: Determine what transformations have the correct insert from Aug. 4, 2010.
Method: Colony PCR. Changes - Used pipette tip instead of toothpick.
PCR: Thermocycler set to iGEM PFUTEST

1. pLacI-mRBS: 4 (A - E) 50(µL) 2. pBAD-mRBS: 5 (A - E) 20(µL) 3. mRBS-TetR: 6 (A -E) 20(µL) Controls - mRBS

Component 1X(µL) Master Mix(x7.5)(µL)
Milli-Q H₂O 9.8 73.5
10x Pfu Buffer with MgSO₄ 2 15
dNTPs 2 15
Forward Primer (VF2) 2 15
Reverse Primer (VR) 2 15
Template DNA 2
Pfu polymerase 0.2 1.5

Added 18µL Master Mix to each reaction tube.

(In Lab: ADS)

Objective: Transform mRBS-xylE BioBrick into DH5alpha.

Transformation -
A) Thawed 50(µL) Sub-Cloning Efficiency DH5alpha Competent Cells on ice. B) Gently mixed cells and then aliquoted 100(µL) into chilled polypropylene tubes. C) Added 2(µL) of BioBrick to cells. Added 5(µL) of pUC19 DNA to 100(µL) cells to determine efficiency. D) Incubated cells on ice for 30 minutes. E) Heat shocked cells for 45 seconds in a 42^oC water bath. F) Placed on ice for 5 minutes. G) Added 0.4 mL of room temperature SOC medium. H) Shook at 225 rpm for 1 hour. I) Diluted control cells 1:100 with SOC medium. J) Spread 100(µL) of this dilution on LB-Amp agar plates K) Spread 50 and 250(µL) of experimental cells on LB-Cam agar plates. L) Incubated overnight at 37^oC

Aug 12, 2010

(In Lab: JV)

Objective: Determine if Adam's colony PCRs' from Aug. 11, 2010 worked.
Method: Samples were run on a 2.5% agarose gel (1X TAE) for 1 hour at 100V.

GEL PICTURE!
Results: Lanes 2, 3, 4 and 8 showed PCR amplification. Colonies chosen don't show the correct insert size.

(In Lab: JV)
Objective: Screen for colonies with the correct insert from Aug. 4, 2010 transformations.

Method: Colony PCR. Changes - Used pipette tip instead of toothpick. Put colony in 20(µL) autoclaved Milli-Q water.
PCR: Thermocycler set to iGEM PFUTEST

1. pLacI-sRBS: A (11 - 17) 2. pBAD-sRBS: B (11 - 17) 3. dT-pTet: C (11 - 17) 4. pLacI-mRBS: D (11-17) 5. pBAD-mRBS: E (11-17) 6. mRBS-TetR: F (11-17)
Controls - mRBS, pTet, sRBS

Component 1X(µL) Master Mix(x47)(µL)
Milli-Q H₂O 9.8 460.6
10x Pfu Buffer with MgSO₄ 2 94
dNTPs 2 94
Forward Primer (VF2) 2 94
Reverse Primer (VR) 2 94
Template DNA (Cell Lysate) 2 94
Pfu polymerase 0.2 9.4

Added 18µL Master Mix to each reaction tube. Analyzed PCR products on 2.5% TAE gel. GEL PICTURE!!!

(In Lab: ADS, KG)
Objective: Perform PCR on lumazine, mms6, xylE plasmids with prefix and suffix primers (these will tell us exact size without subtracting VF2/VR regions). If right will ligate into pET28a plasmids.

Method: Plasmids used included: 6 mms6 maxipreps. 4 lumazine maxipreps. 5 xylE maxipreps. 1 mRBS maxiprep
PCR: Thermocycler set to iGEM Program #11 PFU - P/S

PCR- Conditions: 1. Initial Denaturation 95^oC for 3 min 2. Denaturation 95^oC for 30 sec 3. Anneal (54^oC) for 30 sec 4. Extend 72^oC for 3 min 5. Final Extend 72^oC for 15 min 6. Held 4^oC infinitely (25 cycles)

Component 1X(µL) Master Mix(x16.5)(µL)
Milli-Q H₂O 9.8 161.7
10x Pfu Buffer with MgSO₄ 2 33
dNTPs 2 33
Forward Primer (Prefix) 2 33
Reverse Primer (Suffix Antisense) 2 33
Template DNA (Cell Lysate) 2
Pfu polymerase 0.2 3.3

Added 18µL Master Mix to each reaction tube. Analyzed PCR products on 2.5% TAE gel run at 100 V for 35 minutes. GEL PICTURE!!!

Aug 13, 2010

(In Lab: AS)

Objective: PCR amplify minipreps prepared on Aug 9/2010 to screen for properly assembled BioBricks.

Component 1X(µL) Master Mix(x12.5)(µL)
Milli-Q H₂O 41.85 523.1
10x Pfu Buffer with MgSO₄ 5 62.5
dNTPs 1 12.5
Forward Primer (VF2) 0.5 6.25
Reverse Primer (VR) 0.5 6.25
Template DNA 1
Pfu polymerase 0.15 1.888

Added 49µL Master Mix to each reaction tube.

(In Lab: AS)

Objective: PCR amplify pSB1A3, pSB1T3 and pSB1C3 for use in future 3 part assembly and subsequent growth for glycerol stock.

Component 1X(µL) Master Mix(x13.5)(µL)
Milli-Q H₂O 14.9 52.15
10x Pfu Buffer with MgSO₄ 2 7
dNTPs 1 3.5
Forward Primer (SB-prep-2) 0.7 2.45
Reverse Primer (SB-prep-3p) 0.7 2.45
Template DNA 0.5
Pfu polymerase 0.2 0.7

PCR- Conditions: 1. 94^oC for 30 sec 2. 94^oC for 30 sec 3. 55^oC for 30 sec 4. 68^oC for 3 min 5. 68^oC for 10 min 6. 4^oC infinitely (36 cycles)
Added 19.5µL Master Mix to each reaction tube. Analyzed products on 1% TAE agarose gel which ran for 60 minutes at 100 V.
GEL PICTURE!

Aug 14, 2010

(In Lab: AS)

2.5% agarose gel(1x TAE)

lane contents
1 pBAD-mRBS 1
2 pBAD-mRBS 2
3 pBAD-sRBS 1
4 pBAD-sRBS 2
5 mRBS-TetR 1
6 mRBS-TetR 3
7 50 bp Ladder
8 dT-pTet 1
9 dT-pTet 3
10 pLacI-mRBS 1
11 pLacI-mRBS 2
12 pLacI-sRBS 2
13 pLacI-sRBS 3
14 MT
15 MT
16 MT
17 MT
18 MT
19 MT
20 MT
21 No Lanes
22 No Lanes
23 No Lanes
24 No Lanes
25 No Lanes
26 No Lanes
27 No Lanes

lane contents
1 K249001
2 K249004
3 K249005
4 K249006
5 MT
6 K249008
7 K249008 (Qiagen)
8 K249014
9 K249017
10 50 bp Ladder
11 1
12 2
13 3
14 4
15 5
16 xylE-dT
17 Lumazine-dT
18 pLacI-sRBS
19 MT
20 MT
21 MT
22 MT
23 MT
24 MT
25 MT
26 MT
27 MT

GEL PICTURE!

(In Lab: AS)

Objective: Insert mms6 and lumazine into pET28a using NotI restriction site.

Method: 1. Reserve 1(µL) of dirty PCR product for analysis. 2. Clean up PCR products using Qiagen prep. 3. Restrict 4 mms6 maxipreps and 4 lumazine maxipreps.

Restriction Reactions:
For lumazine and mms6 -

Ingredient 1X(µL) Master Mix(x8.5)(µL)
MilliQ H₂0 Water 7.8 66.30
Orange Buffer (10x) 2 17
pDNA 10
NotI 0.2 1.7

Added 10 (µL) to each tube.
For pET28a -

Ingredient Reaction Mix(µL)
MilliQ H₂0 Water 4.8
Orange Buffer (10x) 5
pDNA 40
NotI 0.2

Incubated at 37^oC. Analyzed results on 2.5% TAE agarose gel which ran at 100 V for 50 minutes. GEL PICTURE!!!
Results: Lost all the DNA in the column clean-up step and will have to re-do.

Aug 14, 2010 Evening

(In Lab: AS)

Objective: Assemble mms6-dT and lumazine-dT using three antibiotic assembly.

Method: 1. PCR amplify BioBricks (Prefix/Suffix) 2. Restrict BioBricks 3. Ligate BioBricks into psB1C3 4. Confirm ligation by PCR analysis (VF2/VR) 5. Transform ligation mixes 6. Screen colonies with Colony PCR

PCR: Thermocycler set to iGEM program 11

Component 1X(µL) Master Mix(x10.5)(µL)
Milli-Q H₂O 10.8 113.4
10x Pfu Buffer with MgSO₄ 2 21
dNTPs 2 21
Forward Primer (Prefix) 2 21
Reverse Primers (Suffix Antisense) 2 21
Template DNA 1
Pfu polymerase 0.2 2.1

Added 19 (µL) to each tube.
Restriction Reactions:
For lumazine and mms6 -

Ingredient 1X(µL) Master Mix(x5.5)(µL)
MilliQ H₂0 Water 11.6 63.8
Red Buffer (10x) 2 11
pDNA 6
EcoRI 0.2 1.1
SpeI 0.2 1.1

Added 14 (µL) to each tube.
For dT -

Ingredient Reaction Mix(µL)
MilliQ H₂0 Water 58
Tango Buffer (10x) 10
pDNA 30
XbaI 1
PstI 1

For psB1C3 -

Ingredient Reaction Mix(µL)
MilliQ H₂0 Water 70
Orange Buffer (10x) 10
pDNA 30
EcoRI 1
PstI 1
DpnI 1

Also cut lumazine, mms6 and dT with one enzyme for two part, PCR amplification and subsequent ligation into pSB1X3.
For lumazine and mms6, CUT with SpeI -

Ingredient 1X(µL) Master Mix(x5.5)(µL)
MilliQ H₂0 Water 15.8 86.9
Tango Buffer (10x) 2 11
pDNA 2
SpeI 0.2 1.1

Added 18 (µL) to each reaction.
For dT, CUT with XbaI-

Ingredient Reaction Mix(µL)
MilliQ H₂0 Water 79
Tango Buffer (10x) 10
pDNA 10
XbaI 1

Incubated at 37^oC for 1.5 hours. Heat killed enzymes for 20 minutes at 80^oC.
Ligation Reactions:
3 Part: Lumazine/mms6 + dT + psB1C3

Ingredient 1X(µL) Master Mix(x5.5)(µL)
MilliQ H₂0 Water 11.0 64.9
T4 Ligase Buffer (10x) 2 11
Plasmid (psB1C3) 2 11
Part 1 (Lumazine/mms6) 2
Part 2 (dT) 2 11
T4 DNA Ligase 0.2 1.1

2 Part: Lumazine/mms6 + dT

Ingredient 1X(µL) Master Mix(x5.5)(µL)
MilliQ H₂0 Water 13.8 75.9
T4 Ligase Buffer (10x) 2 11
Part 1 (Lumazine/mms6) 2
Part 2 (dT) 2 11
T4 DNA Ligase 0.2 1.1

Added 18(µL) to each rxn tube. Incubated 1 hour and overnight at room temperature ( 25^oC).

Screening via PCR amplification : Thermocycler set to iGEM program 11
3 Part: Lum/mms6 + dT + pSB1C3

Component 1X(µL) Master Mix(x5.5)(µL)
Milli-Q H₂O 33.8 185.9
10x Pfu Buffer with MgSO₄ 5 27.5
dNTPs 2 11
VF2 Primer 2 11
VR Primer 2 11
Template DNA 5
Pfu polymerase 0.2 1.1

Added 45 (µL) MM to each tube.
2 Part: Lum/mms6 + dT

Component 1X(µL) Master Mix(x5.5)(µL)
Milli-Q H₂O 6.8 37.4
10x Pfu Buffer with MgSO₄ 2 11
dNTPs 2 11
Prefix Primer 2 11
Suffix Antisense Primer 2 11
Template DNA 5
Pfu polymerase 0.2 1.1

Added 15 (µL) MM to each tube.

Aug 15, 2010

(In Lab: ADS)

Objective: Insert mms6 and lumazine into pET28a using NotI restriction site.

Method: 1. Reserve 1(µL) of dirty PCR product for analysis. 2. Clean up PCR products using Qiagen prep. 3. Restrict 3 mms6 maxipreps and 2 lumazine maxipreps.

Restriction Reactions:
For lumazine and mms6 -

Ingredient 1X(µL) Master Mix(x10.5)(µL)
MilliQ H₂0 Water 11.8 123.9
Orange Buffer (10x) 2 21
pDNA 6
NotI 0.2 2.1

Added 14 (µL) to each tube.
For pET28a -

Ingredient Reaction Mix(µL)
MilliQ H₂0 Water 4.8
Orange Buffer (10x) 5
pDNA 40
NotI 0.2

Incubated at 37^oC for 1.5 hours. Heat killed enzymes at 80^oC for 20 minutes.
Ligation Reactions:

Ingredient 1X(µL) Master Mix(x5.5)(µL)
MilliQ H₂0 Water 13.9 75.9
T4 Ligase Buffer (10x) 2 11
Plasmid (pET28a) 2 11
Cut out part (Lumazine/mms6) 2
T4 DNA Ligase 0.2 1.1

Added 18(µL) to each tube. Incubated 1 hour and overnight at room temperature.

Aug 15, 2010 Evening

(In Lab: AS)

Objective: Assemble Lum-dT & mms6-dT using BioBrick standard assembly.

Method: Obtain plasmid DNA from maxipreps.

Restriction Reactions:
For lumazine and mms6 -

Ingredient 1X(µL) Master Mix(x5.5)(µL)
MilliQ H₂0 Water 7.6 41.8
Red Buffer (10x) 2 11
pDNA 10
EcoRI 0.2 1.1
SpeI 0.2 1.1

For dT -

Ingredient Reaction Mix(µL)
MilliQ H₂0 Water 38
Orange Buffer (10x) 10
pDNA 50
XbaI 1
EcoRI 1

Incubated at 37^oC for 1.5 hours. Heat killed enzymes for 20 minutes at 80^oC.
Ligation Reactions:

Ingredient 1X(µL) Master Mix(x5.5)(µL)
MilliQ H₂0 Water 13.8 75.9
T4 Ligase Buffer (10x) 2 11
Plasmid (dT) 2 11
Cut out part (Lumazine/mms6) 2
T4 DNA Ligase 0.2 1.1

Added 18(µL) MM to each rxn tube. Incubated at one hour and overnight at room temperature.
Screening via PCR amplification : Thermocycler set to iGEM program 11

Component 1X(µL) Master Mix(x5.5)(µL)
Milli-Q H₂O 36.8 185.9
10x Pfu Buffer with MgSO₄ 5 27.5
dNTPs 2 11
VF2 Primer 2 11
VR Primer 2 11
Template DNA 2
Pfu polymerase 0.2 1.1

Added 45 (µL) MM to each tube.
Analyzed PCR products of BioBrick standard assembly; 3 part (or 3 antibiotic) assembly; and 3 part (3AB) Intermediate/2 part assembly on a 2% TAE agarose gel.
GEL PICTURE!

(In Lab: ADS)

Objective: Produce large quantities of pSB1A3, pSB1C3 and pSB1T3.

Method:

Component 1X(µL) Master Mix(x3.2)(µL)
Milli-Q H₂O 75 240
10x Pfu Buffer with MgSO₄ 10 32
dNTPs 5 16
Primer (SB-prep-2Ea) 3.5 11.2
Primer (SB-prep-3P) 3.5 11.2
Template DNA 2
Pfu polymerase 1 3.2

Added 98(µL) to each tube. Used PLASRB PCR Protocol from Aug. 13, 2010.

Aug 16, 2010

(In Lab: KG)

Objective: Confirm overnight ligations done on August 15, 2010.

Method:

Screening via PCR amplification : Thermocycler set to iGEM program 4

Component 1X(µL)
Milli-Q H₂O 33.8
10x Pfu Buffer with MgSO₄ 5
dNTPs 2
VF2 Primer 2
VR Primer 2
Template DNA 5
Pfu polymerase 0.2

3AB Master Mix

Component Master Mix(x5.5)(µL)
Milli-Q H₂O 185.9
10x Pfu Buffer with MgSO₄ 27.5
dNTPs 11
Prefix Primer 11
Suffix Primer 11
Template DNA 2
Pfu polymerase 1.1

BBS/pSB1C3 Master Mix

Component Master Mix(x11)(µL)
Milli-Q H₂O 371.8
10x Pfu Buffer with MgSO₄ 55
dNTPs 22
VF2 Primer 22
VR Primer 22
Template DNA
Pfu polymerase 2.2

Analyzed PCR products of overnight BioBrick standard assembly; 3 part (or 3 antibiotic) assembly; and 3 part (3AB) Intermediate/2 part assembly on a 2% TAE agarose gel.
GEL PICTURE!

Aug 16, 2010 Evening

(In Lab: KG, AS)

Objective: Restriction of PCR Products (mms6-dT, lumazine-dT). Restriction is necessary for ligation into plasmid backbone pSB1C3

Method:

Restriction Reactions:

Ingredient 1X(µL) Master Mix(x5.5)(µL)
MilliQ H₂0 Water 7.6 41.8
Orange Buffer (10x) 2 11
pDNA 10
PstI 0.2 1.1
SpeI 0.2 1.1

Incubated at 37^oC for 1.5 hours. Heat killed enzymes for 2 minutes at 80^oC.
Ligation Reactions:

Ingredient 1X(µL) Master Mix(x5.5)(µL)
MilliQ H₂0 Water 13.8 75.9
T4 Ligase Buffer (10x) 2 11
Plasmid Backbone (pSB1C3) 2 11
pDNA 2
T4 DNA Ligase 0.2 1.1

(In Lab: KG)

Objective: Transformations of insertions of mms6 or lumazine into pET28a.

Method: used Competent Cell Transformation protocol

changes:
used 50µL aliquottes of DH5&alpha
did not pipette up and down once, the cells were just swirled 3 times
added 400µL SOC media, shoock at 37⁰C for 90 min
platted 250µL and 150µL

results
contents &250µL 150µL
+ control(pUC19) good good
mms6 good good
mms6-2 good good
mms6 good x
Lumazine good x
Lumazine good x

Aug 17, 2010

(In Lab: JV)

Objective: Confirm ligations done on August 16, 2010.

Method:
Screening via PCR amplification : Thermocycler set to iGEM program 4

Component 1X(µL) Master Mix(x5.5)(µL)
Milli-Q H₂O 12.8 70.4
10x Pfu Buffer with MgSO₄ 2 11
dNTPs 1 5.5
VF2 Primer 1 5.5
VR Primer 1 5.5
Template DNA 2
Pfu polymerase 0.2 1.1

Ran a 2% Agarose gel in 1X TAE buffer for 65 minutes at 100V.
GEL PICTURE!

Aug 17, 2010 Evening

(In Lab: AS)

Objective: Repeat ligation of mms6-dT and lumazine-dT to pSB1C3.
Method: Use already restricted mms6-dT and lumazine-dT. Restrict pSB1C3 PCR product with EcoRI and PstI.

Restriction Reactions:

Ingredient 1X(µL)
MilliQ H₂0 Water 28.6
Orange Buffer (10x) 6
pDNA (pSB1C3) 25
PstI 0.2
EcoRI 0.2

Incubated at 37^oC for 1.5 hours. Heat killed enzymes for 20 minutes at 80^oC.
Ligation Reactions:

Ingredient 1X(µL) Master Mix(x5.5)(µL)
MilliQ H₂0 Water 13.8 75.9
T4 Ligase Buffer (10x) 2 11
Part 2 (pSB1C3) 2 11
Part 1 (mms6-dT/Lum-dT) 2
T4 DNA Ligase 0.2 1.1

Added 18(µL) MM to each rxn tube. Incubated overnight at room temperature.

Objective: Ligation confirmation by PCR. 2 different PCR reaction conditions were utilized. Believe PstI is not being heat inactivated.

Method:
PCR 1 - Show complete insertion of mms6-dT, lumazine-dT into pSB1C3. Both EcoRI and PstI ligations occurred.

Component 1X(µL) Master Mix(x11)(µL)
Milli-Q H₂O 33.8 371.8
10x Pfu Buffer with MgSO₄ 5 55
dNTPs 2 22
Forward VF2 Primer 2 22
Reverse VR Primer 2 22
Template DNA 5
Pfu polymerase 0.2 2.2

Added 45(µL) MM to each rxn tube.
PCR 2 - Show PstI is not heat killed and only EcoRI ligation occurred.

Component 1X(µL) Master Mix(x16)(µL)
Milli-Q H₂O 33.8 540.8
10x Pfu Buffer with MgSO₄ 5 80
dNTPs 2 32
Forward VF2 Primer 2 32
Reverse VR Primer 2 32
Template DNA 5
Pfu polymerase 0.2 3.2

Added 45(µL) MM to each rxn tube.

Aug 19, 2010

(In Lab: JV)

Objective: Determine if any transformations from Aug 16, 2010 have the correct insert.
Method: Pick colonies and incubate at 37^oC in LB Media with Kan overnight. Use QIAGEN method to extract plasmid DNA. Restrict plasmid DNA to determine if mms6 or lumazine has correctly ligated into pET-28(A).

Restriction Reactions:
mms6 RESTRICTED -

Ingredient 1X(µL) Master Mix(x31)(µL)
MilliQ H₂0 Water 15.75 488.25
Red Buffer (10x) 2 62
Template DNA
Enzyme (EcoRV) 0.25 7.75

mms6 UNRESTRICTED -

Ingredient 1X(µL) Master Mix(x31)(µL)
MilliQ H₂0 Water 16 496
Red Buffer (10x) 2 62
Template DNA

lumazine RESTRICTED -

Ingredient 1X(µL) Master Mix(x31)(µL)
MilliQ H₂0 Water 15.75 488.25
Tango Buffer (10x) 2 62
Template DNA
Enzyme (EcoRV) .25 7.75

lumazine UNRESTRICTED -

Ingredient 1X(µL) Master Mix(x31)(µL)
MilliQ H₂0 Water 16 496
Tango Buffer (10x) 2 62
Template DNA

Added 18(µL) to each restriction digest reaction. Incubated at 37^oC for 1.5 hours. Heat killed enzymes for 20 minutes at 80^oC.
Samples were run on a 2% agarose gel in 1X TAE Buffer.
GEL PICTURE!

Aug 20, 2010

(In Lab: JV)

Objective: Determine if attempts to PCR amplify plasmid backbone were successful.

Method: Ran samples on 1% agarose gel with 1X TAE buffer for 50 minutes at 100 V.

Results: DNA concentration was good, however there was no evidence of an insert into pET-28(A).

GEL PICTURE!!!

Aug 23, 2010

(In Lab: JV)

Objective: Obtained part <partinfo>BBa_K118021</partinfo> and <partinfo>BBa_I716462</partinfo>.

Method: Used competent cell transformation protocol.

(In Lab: HB)

Objective: Restrict 18 maxiprepped parts to quantify DNA.

Method: Restrict all 18 parts and run on a 1% agarose gel with unrestricted parts.

Restriction Reactions:
Restriction Mix -

Ingredient 1X(µL) Master Mix(x19)(µL)
MilliQ H₂0 Water 12.8 243.2
Orange Buffer (10x) 2 38
Template DNA 5
EcoRI 0.2 3.8

Unrestricted Mix -

Ingredient 1X(µL) Master Mix(x19)(µL)
MilliQ H₂0 Water 13 247
Orange Buffer (10x) 2 38
Template DNA 5

15(µL) added to each rxn tube. Incubated at 37^oC for 1.5 hours.
GEL PICTURE!
Retrieved from "http://2010.igem.org/Team:Lethbridge/Notebook/Lab_Work/August"

@@ Line 1,366: / Line 1,366: @@
 GEL PICTURE!
+----
+(In Lab: ADS)<br>
+<b>Objective:</b> Produce large quantities of pSB1A3, pSB1C3 and pSB1T3.<br>
+<b>Method:</b><br>
+<table border ="3">
+<tr><td><b>Component</b><td><b>1X(&micro;L)</b><td><b>Master Mix(x3.2)(&micro;L)</b>
+<tr><td>Milli-Q H<sub>2</sub>O<td>75<td>240
+<tr><td>10x Pfu Buffer with MgSO<sub>4</sub><td>10<td>32
+<tr><td>dNTPs<td>5<td>16
+<tr><td>Primer (SB-prep-2Ea)<td>3.5<td>11.2
+<tr><td>Primer (SB-prep-3P)<td>3.5<td>11.2
+<tr><td>Template DNA<td>2<td>
+<tr><td>Pfu polymerase<td>1<td>3.2
+</table><br>
+Added 98(&micro;L) to each tube.  Used PLASRB PCR Protocol from Aug. 13, 2010.
 ==<font color="white">Aug 16, 2010 ==

Construct	pDNA	buffer	Enzymes
pBAD-sRBS/mRBS	pBAD	Red	EcoRI and SpeI
pBAD-sRBS/mRBS	sRBS	Orange	XbaI and EcoRI
pBAD-sRBS/mRBS	mRBS	Orange	XbaI and EcoRII
sRBS/mRBS-TetR	sRBS	Red	PstI and SpeI
sRBS/mRBS-TetR	mRBS	Red	PstI and SpeI
sRBS/mRBS-TetR	TetR	Tango	XbaI and PstI
TetR-dT	TetR	Red	EcoRI and SpeI
TetR-dT	dT	Orange	XbaI and EcoRI
dT-pTet	dT	Red	EcoRI and SpeI
dT-pTet	pTet	Orange	XbaI and EcoRI
pLAcI-sRBS/mRBS	pLacI	Red	EcoRI and SpeI
pLAcI-sRBS/mRBS	sRBS	Orange	XbaI and EcoRI
pLAcI-sRBS/mRBS	mRBS	Orange	XbaI and EcoRI
Mms6-PET28(a)	PET28(a)	Orange	NotI

Component	Volume per tube (µL)	Master Mix (x6)
MilliQ H₂O	41.8	250.8
10x Pfu Buffer with MgSO₄	5	30
dNTPs	1	6
Forward Primer	0.5	3
Reverse Primer	0.5	3
Template DNA	1	-
Pfu DNA polymerase	0.2	-
Total	50	292.8

Lane	Sample	Components (µL)
1	1 kb ladder	2 loading dye (5x) + 0.5 ladder + 3.5 MilliQ H₂O
2	ECFP	2 DNA + 2 loading dye (5x) + 2 MilliQ H₂O
3	EYFP	2 DNA + 2 loading dye (5x) + 2 MilliQ H₂O
4	Lumazine	2 DNA + 2 loading dye (5x) + 2 MilliQ H₂O

Restriction	MilliQ H₂O (µL)	Buffer Orange (µL)	pDNA (µL)	Enzyme (µL)
pSB1C3	79.25	10	10	0.25 EcoRI + 0.25 PstI + 0.25 DpnI
pSB1C3 control	80	10	10	-
dT	79.50	10	10	0.25 EcoRI + 0.25 PstI
dT control	80	10	10	-

Lane	Contents	Result
1	1kb ladder
2	pTet (XbaI, EcoRI)	good
3	pTet (orange buffer)	---
4	dT (XbaI, EcoR)	no good
5	dT (orange buffer)	---
6	mRBS (SpeI, PstI)	good
7	mRBS (red buffer)	---
8	sRBS (SpeI, PstI)	good
9	sRBS (red buffer)	---
10	mRBS (XbaI, EcoRI)	good
11	mRBS (orange buffer)	---
12	sRBS (XbaI, EcoRl)	good
13	sRBS (orange buffer)	---
14	pet28(a)	good
15	100 bp ladder
16	PSB1C3	not good
17	PSB1C3 restriction digest	---

Component	1X(µL)	Master Mix(x16)(µL)
Milli-Q H₂O	41.8	668.6
10x Pfu Buffer with MgSO₄	5	80
dNTPs	1	16
Forward Primer	0.5	8
Reverse Primers	0.5	8
Template DNA	1	16
Pfu polymerase	0.2	3.2

lane	contents	Successful Ligation ?
1	50bp ladder	---
2	dt pSBIC3	---
3	dt pTet	x
4	dt control	---
5	sRBS-TetR	x
6	mRBS-TetR	?
7	TetR control	---
8	pLacI-mRBS	x
9	pLacI-sRBS	?
10	pLacI control	---
11	Mms6 pET-28a	no band
12	Mms6 control	---
13	pBad-SRBS	x
14	pBad-mRBS	x
15	pBad control	---

results
contents	&250µL	150µL
dt-pTet	good	x
- control	x	x
mms6-pET-28a	good	good
dt-pSBIC3	x	x
mRBS-TetR	good	good
pLacI-mRBS	good	good
SRBS-TetR	x	x
pBAD-SRBS	good	good
+ contol	good	good

lane	sample	loaded
1	mms6 restricted with NotI	1 mL sample
2	mms6 unrestricted control	10(µL) sample, 2(µL)dye
3	50bp ladder	2(µL) ladder, 2(µL) dye, 8(µL) H₂0
3	1 kb ladder	2(µL) ladder, 2(µL) dye, 8(µL) H₂0

Knight cont'd	Endy cont'd
setup 20(µL) reaction	setup 20(µL) reaction
1(µL) colony suspension	2(µL) colony suspension
2(µL) 10x p.fu (+Mg SO₄	2(µL) 10x p.fu (+Mg SO₄
2(µL) dNTP	2(µL) dNTP
1.25(µL)VF2 Primer (10(µM)	0.75(µL)VF2 Primer (10(µM)
1.25(µL)VF Primer (10(µM)	0.75(µL)VF Primer (10(µM)
0.2(µL) Pfu polymerase	0.2(µL) Pfu polymerase
11.8 Milli-Q H₂O	12.6 Milli-Q H₂O

Knight cont'd	Endy cont'd
cycling conditions	cycling conditions
95⁰C for 15 minutes	95⁰C for 6 minutes
*94⁰C for 30 seconds	**95⁰C for 30 seconds
*56⁰C for 30 seconds	**56⁰C for 30 seconds
*68⁰C for 1 minutes	**70⁰C for 1 minutes
68⁰C for 20 minutes	70⁰C for 10 minutes

Component	1X(µL)	Master Mix(x4)(µL)
Milli-Q H₂O	41.8	167.2
10x Pfu Buffer with MgSO₄	5	20
dNTPs	1	4
Forward Primer (VF2)	0.5	2
Reverse Primers (VR)	0.5	2
Template DNA	1
Pfu polymerase	0.2

Ingredient	Volume(µL)
MilliQ H₂0 Water	15.75
Orange Buffer (10x)	2
pDNA	2
EcoRI	0.25

Component	Volume (µL)
MilliQ H₂O	15.6 or 15.8
Buffer	2
pDNA	2
Enzyme	0.20 + 0.20

Lane	Contents
1	50 bp ladder
2	pBAD-mRBS 2 URD
3	pBAD-mRBS 2 SRD
4	pBAD-mRBS 2 DRD
5	pLacI-sRBS 3 URD
6	pLacI-sRBS 3 SRD
7	pLacI-sRBS 3 DRD
8	pLacI-mRBS 2 URD
9	pLacI-mRBS 2 SRD
10	pLacI-mRBS 1 DRD
11	dT-pTet 3 URD
12	dT-pTet 3 SRD
13	dT-pTet 3 DRD
14	pBAD-mRBS 1 URD
15	pBAD-mRBS 1 SRD
16	pBAD-mRBS 1 DRD
17	pLacI-sRBS 2 URD
18	pLacI-sRBS 2 SRD
19	pLacI-sRBS 2 DRD
20	1 kb ladder

Component	1X(µL)	Master Mix(x65)(µL)
Milli-Q H₂O	10.9	708.5
5X Phusion HF Buffer	4	260
dNTPs	1	65
Forward Primer (VF2)	1	65
Reverse Primers (VR)	1	65
Colony Template	2
Phusion polymerase	0.17	11.05

Component	1X(µL)	Master Mix(x6.5)(µL)
Milli-Q H₂O	8.8	57.2
5X Phusion HF Buffer	4	26
dNTPs	2	13
Forward Primer	2	13
Reverse Primer	2	13
Template DNA	1	6.5
Polymerase	0.2	1.3

Component	1X(µL)	Master Mix(x7.5)(µL)
Milli-Q H₂O	9.8	73.5
10x Pfu Buffer with MgSO₄	2	15
dNTPs	2	15
Forward Primer (VF2)	2	15
Reverse Primer (VR)	2	15
Template DNA	2
Pfu polymerase	0.2	1.5

Component	1X(µL)	Master Mix(x47)(µL)
Milli-Q H₂O	9.8	460.6
10x Pfu Buffer with MgSO₄	2	94
dNTPs	2	94
Forward Primer (VF2)	2	94
Reverse Primer (VR)	2	94
Template DNA (Cell Lysate)	2	94
Pfu polymerase	0.2	9.4

Component	1X(µL)	Master Mix(x16.5)(µL)
Milli-Q H₂O	9.8	161.7
10x Pfu Buffer with MgSO₄	2	33
dNTPs	2	33
Forward Primer (Prefix)	2	33
Reverse Primer (Suffix Antisense)	2	33
Template DNA (Cell Lysate)	2
Pfu polymerase	0.2	3.3

Component	1X(µL)	Master Mix(x12.5)(µL)
Milli-Q H₂O	41.85	523.1
10x Pfu Buffer with MgSO₄	5	62.5
dNTPs	1	12.5
Forward Primer (VF2)	0.5	6.25
Reverse Primer (VR)	0.5	6.25
Template DNA	1
Pfu polymerase	0.15	1.888

Component	1X(µL)	Master Mix(x13.5)(µL)
Milli-Q H₂O	14.9	52.15
10x Pfu Buffer with MgSO₄	2	7
dNTPs	1	3.5
Forward Primer (SB-prep-2)	0.7	2.45
Reverse Primer (SB-prep-3p)	0.7	2.45
Template DNA	0.5
Pfu polymerase	0.2	0.7

lane	contents
1	pBAD-mRBS 1
2	pBAD-mRBS 2
3	pBAD-sRBS 1
4	pBAD-sRBS 2
5	mRBS-TetR 1
6	mRBS-TetR 3
7	50 bp Ladder
8	dT-pTet 1
9	dT-pTet 3
10	pLacI-mRBS 1
11	pLacI-mRBS 2
12	pLacI-sRBS 2
13	pLacI-sRBS 3
14	MT
15	MT
16	MT
17	MT
18	MT
19	MT
20	MT
21	No Lanes
22	No Lanes
23	No Lanes
24	No Lanes
25	No Lanes
26	No Lanes
27	No Lanes

lane	contents
1	K249001
2	K249004
3	K249005
4	K249006
5	MT
6	K249008
7	K249008 (Qiagen)
8	K249014
9	K249017
10	50 bp Ladder
11	1
12	2
13	3
14	4
15	5
16	xylE-dT
17	Lumazine-dT
18	pLacI-sRBS
19	MT
20	MT
21	MT
22	MT
23	MT
24	MT
25	MT
26	MT
27	MT

Ingredient	1X(µL)	Master Mix(x8.5)(µL)
MilliQ H₂0 Water	7.8	66.30
Orange Buffer (10x)	2	17
pDNA	10
NotI	0.2	1.7

Ingredient	Reaction Mix(µL)
MilliQ H₂0 Water	4.8
Orange Buffer (10x)	5
pDNA	40
NotI	0.2

Component	1X(µL)	Master Mix(x10.5)(µL)
Milli-Q H₂O	10.8	113.4
10x Pfu Buffer with MgSO₄	2	21
dNTPs	2	21
Forward Primer (Prefix)	2	21
Reverse Primers (Suffix Antisense)	2	21
Template DNA	1
Pfu polymerase	0.2	2.1

Ingredient	1X(µL)	Master Mix(x5.5)(µL)
MilliQ H₂0 Water	11.6	63.8
Red Buffer (10x)	2	11
pDNA	6
EcoRI	0.2	1.1
SpeI	0.2	1.1

Component	1X(µL)	Master Mix(x3.2)(µL)
Milli-Q H₂O	75	240
10x Pfu Buffer with MgSO₄	10	32
dNTPs	5	16
Primer (SB-prep-2Ea)	3.5	11.2
Primer (SB-prep-3P)	3.5	11.2
Template DNA	2
Pfu polymerase	1	3.2

Component	Master Mix(x11)(µL)
Milli-Q H₂O	371.8
10x Pfu Buffer with MgSO₄	55
dNTPs	22
VF2 Primer	22
VR Primer	22
Template DNA
Pfu polymerase	2.2