Contents 1 September 1.1 September 2, 2010 1.1.1 ADS 1.2 September 14, 2010 1.2.1 JF 1.2.2 JV 1.2.3 KG 1.2.4 AS 1.3 September 15, 2010 1.3.1 JV, ADS 1.4 September 16, 2010 1.4.1 JV 1.5 September 17, 2010 1.5.1 JV 1.5.2 AV 1.5.3 KG 1.6 September 18, 2010 1.6.1 JS, DM 1.6.2 TF 1.7 September 20, 2010 1.7.1 ADS 1.7.2 HB 1.8 September 21, 2010 1.8.1 ADS 1.8.2 TF, MC 1.9 September 21, 2010 1.9.1 ADS 1.10 September 23, 2010 1.10.1 JV 1.10.2 ADS 1.10.3 TF, MC 1.11 September 24, 2010 1.11.1 ADS 1.12 September 25, 2010 1.12.1 JV 1.12.2 ADS 1.13 September 26, 2010 1.13.1 ADS 1.14 September 29, 2010 1.14.1 ADS

September

September 2, 2010

ADS

Objective: Assemble lumazine-dT into pSB1C3 using NEB BioBrick Assembly Kit.
Method: Have the following parts: dT [28 ng/(µL)]; lumazine [78 ng/(µL)]; psB1C3 [14 ng/(µL)].

• Require 500 ng DNA each part in a 50(µL) rxn therefore 17.9(µL) dT and 6.4(µL) Lumazine. Need 250 ng pSB1C3 or 17.9(µL)

Restriction Reactions -

Ingredient	Volume(µL)
MilliQ H20 Water	36.1
NEBuffer 2 (10x)	5
Upstream Part (Lumazine)	26.4
EcoRI-HF	1
SpeI	1
100X BSA	0.5

Ingredient	Volume(µL)
MilliQ H20 Water	24.6
NEBuffer 2 (10x)	5
Downstream Part (dT)	17.9
XbaI	1
PstI	1
100X BSA	0.5

Ingredient	Volume(µL)
MilliQ H20 Water	3.35
NEBuffer 2 (10x)	2.5
Destination Plasmid (pSB1C3)	17.9
EcoRI-HF	.5
PstI	.5
100X BSA	0.25

• 2(µL) was removed prior to adding enzymes for analysis on gel.
  • Split digestion reactions in half: dT - 2 @ 24(µL); Lum - 2 @ 24(µL); psB1C3 - 2 @ 11.5(µL)
    • Ran 10 minute and 60 minute reactions at 37^oC. Followed by 20 minute heat shock at 80^oC.
      • 2(µL) was removed from the heat killed reactions for analysis on gel.

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• Performed 4 combinations of ligation: 10 min Restriction + 10 min Ligation; 10 min Restriction + Overnight Ligation; 60 min Restriction + 10 min Ligation; 60 min Restriction + Overnight Ligation

Ingredient	1X(µL)	2X Master Mix(x2.5)(µL)
MilliQ H20 Water	11	77.5
T4 Ligase Buffer (10x)	2	5
Upstream Part	2	5
Downstream Part	2	5
T4 DNA Ligase	1	2.5
Plasmid Part	2	5

Incubated 10 minute and overnight ligations at room temperature ( 25^oC). Heat killed ligase at 80^oC for 20 min.

• 2(µL) of each reaction was taken for analysis on gel.

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• Confirmed ligation with PCR analysis. Analyzed restriction, ligation and PCR on a 1.5% TAE agarose gel.

Component	1X(µL)	Master Mix(x5.5)(µL)
Milli-Q H2O	36.5	202.4
10x Pfu Buffer with MgSO4	5	27.5
dNTPs	2	11
Forward VF2 Primer	2	11
Reverse VR Primer	2	11
Template DNA	2
Pfu polymerase	0.2	1.1

Added 48(µL) to each reaction.

GEL PICTURE!!!

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Objective: Characterize xylE in LB Media vs. M9 Minimal Media.
Method: Three experiments with spectra.

• Experiment 1 - Measure absorption spectra of 1:10 dilution of: 1. Cells suspended in m9 media +/- catechol. 2. Cells suspended in LB media +/- catechol. 3. m9 media from spun down cells +/- catechol. 4. LB media from spun down cells +/- catechol. 5. Sterile LB media +/- catechol. 6. Water +/- catechol.

• Experiment 2 - Catechol Breakdown. Follow absorbance of 2-hydroxymucanate semialdehyde at 375 nm. Measured all the above samples for 10 min at that wavelength.

September 14, 2010

JF

Objective: Miniprep of RFP expression construct (J04450) via Qiaquick/Qiaprep spin column. Protocol followed as per kit instructions.

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JV

Objective: PCR the pSB1C3 plasmid from part J04450 miniprep.

Component	1X(µL)
Milli-Q H2O	12.8
10x Pfu Buffer with MgSO4	2
dNTPs	1
SB-prep-26 Primer	1
SB-prep-3P Primer	1
Template DNA	2
Pfu polymerase	0.2

Used iGEM cycle PSB1C3 in thermocycler. Ran PCR product on 1% TAE agarose gel.

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KG

Objective: PCR of xylE (mRBS-xylE K118021) with fusion standard so that an arginine tail can be attached.

Component	1X(µL)	Master Mix(x6)(µL)
Milli-Q H2O	12.8	76.8
10x Pfu Buffer with MgSO4	2	12
dNTPs	1	6
xylE Fusion Prefix Forward Primer	1	6
xylE Fusion Suffix Reverse Primer	1	6
Template DNA	2	12
Pfu polymerase	0.2	1.2

PCR- Conditions: 1. 95^oC for 180 sec 2. 95^oC for 30 sec 3. 56.6 +/- 5^oC for 30 sec 4. 72^oC for 150 sec 5. 72^oC for 600 sec (35 cycles)

Analyzed PCR on 2% agarose gel which ran at 100 V for 90 minutes.

AS

Objective: Assemble xylE-dT using 3AB assembly and 3AB assembly.

Restriction Reactions -

3AB Upstream Part (xylE)

Ingredient	Volume(µL)
MilliQ H20 Water	30.6
NEBuffer 2 (10x)	5
Plasmid DNA	11.9
EcoRI-HF	1
SpeI	1
100X BSA	0.5

3AB Downstream Part (dT)

Ingredient	Volume(µL)
MilliQ H20 Water	24.6
NEBuffer 2 (10x)	5
Plasmid DNA	17.9
XbaI	1
PstI	1
100X BSA	0.5

3AB Plasmid (pSB1C3)

Ingredient	Volume(µL)
MilliQ H20 Water
NEBuffer 2 (10x)	5
Plasmid DNA	42.5
EcoRI-HF	1
PstI	1
100X BSA	0.5

• Ran 10 minute and 60 minute reactions at 37^oC. Followed by 20 minute heat shock at 80^oC.

Ligation Reaction -

Ingredient	1X(µL)
MilliQ H20 Water	11
T4 Ligase Buffer (10x)	2
Upstream Part	2
Downstream Part	2
T4 DNA Ligase	1
Plasmid Part	2

Incubated 10 minute and overnight ligations at room temperature ( 25^oC). Heat killed ligase at 80^oC for 20 min.

Transformation -

A) Thawed 200(µL) Library Efficiency DH5alpha Competent Cells on ice. B) Gently mixed cells and then aliquoted 100(µL) into chilled polypropylene tubes. C) Added 1(µL) of ligation mix 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 2 minutes. G) Added 0.9 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

September 15, 2010

JV, ADS

Objective: Determine if dT ligated onto p-rbs-xylE using PCR analysis.

Component	1X(µL)	Master Mix(x4.5)(µL)
Milli-Q H2O	11.8	53.1
10x Pfu Buffer with MgSO4	2	9
dNTPs	1	4.5
Forward VF2 Primer	1	4.5
Reverse VR Primer	1	4.5
Template DNA	1
Pfu polymerase	0.2

Used iGEM program 7 in thermocycler.

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Objective: Determine if minipreps from Sept 15, 2010 worked. Analyze assembly intermediates.
Method: Run 1% TAE agarose gels at 100 V for 90 minutes.

GEL PICTURES!!!

September 16, 2010

JV

Objective: Isolate RFP in pSB1C3 from DH5alpha cells.
Method: Used Qiagen minipep protocol and spin column.
Results: Gel of miniprepped RFP against previously prepared RFP showed a band of the right size.

GEL PICTURE!

September 17, 2010

JV

Objective: Isolate plasmid DNA from DH5alpha cells: K249024; B0015; K118021/B0015
Method: Used Qiagen minipep protocol and spin column. Ran a 1% agarose gel to view DNA

GEL PICTURE!

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AV

Objective: PCR out RFP from the complete construct (J04450) with a N-term fusion standard.

Composition of each PCR tube:

Component	Volume(µL)
10x Pfu Buffer w/ MgSO4	2
dNTP (10mM)	1
N-term Fus Prefix	1
Biobrick Suffix	1
MilliQ H2O	12.8
Template DNA (J04450)	2
Pfu DNA Polymerase	0.2

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KG

Objective: PCR xylE to attach fusion standard to the N-term and standard suffix to C-term.

Component	1X(µL)	Master Mix(x6)(µL)
Milli-Q H2O	12.8	76.8
10x Pfu Buffer with MgSO4	2	12
dNTPs	1	6
Fusion Forward Primer	1	6
Standard Reverse Primer	1	6
Template DNA	2	12
Pfu polymerase	0.2	1.2

Fusion standard prefix has melting temperature of 66.5^oC. Standard suffix has melting temperature of 70.5^oC. Annealing temperature of 61.5^oC with 5 ^oC gradient. PCR- Conditions: 1. 95^oC for 180 sec 2. 95^oC for 30 sec 3. 61.5 +/- 5^oC for 30 sec 4. 72^oC for 150 sec 5. 72^oC for 600 sec (35 cycles)

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Objective: PCR xylE to attach fusion standard to the C-term and standard suffix to N-term.

Component	1X(µL)	Master Mix(x6)(µL)
Milli-Q H2O	12.8	76.8
10x Pfu Buffer with MgSO4	2	12
dNTPs	1	6
Standard Forward Primer	1	6
Fusion Reverse Primer	1	6
Template DNA	2	12
Pfu polymerase	0.2	1.2

Standard prefix has melting temperature of 68.7^oC. Standard suffix has melting temperature of 61.6^oC. Annealing temperature of 56.6^oC with 5 ^oC gradient.

PCR- Conditions: 1. 95^oC for 180 sec 2. 95^oC for 30 sec 3. 56.6 +/- 5^oC for 30 sec 4. 72^oC for 150 sec 5. 72^oC for 600 sec (35 cycles)

September 18, 2010

JS, DM

Objective: Overexpress mms6.
Method: Followed overexpression protocol. One flask was induced with 250(µL) IPTG while another was induced with 50(µL).

Time (minutes)	OD #1 (600λ)	OD #2 (600λ)
30	0.139	0.133
60	0.200	0.188
90	0.372	0.360
120	0.702	0.704
150(T0)	0.871	0.810
210(T1)	2.600	2.500
270(T2)	3.317	3.300
330(T3)	4.408	3.946

• Plated cells in presence of IPTG, Fe or IPTG and Fe on AMP(+) plates. Incubated at 37 ^oC.
  • Preparation of these samples was achieved by taking a 60(µL) cell sample and inoculating 5 mL LB media. These solutions were incubated at 37 ^oC for 30 min. To 1 mL of culture, the appropriate amount of Fe2+ [0.938(µL)], Fe3+[1.88(µL)] or IPTG [1(µL)] was added.

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TF

Objective: Obtain preparations of B0015 (dT) and J04450 (RFP).

Method: Used Plasmid DNA Purification by Alkaline Lysis (Large Scale AKA Maxiprep protocol.

Results: Chloroform, DNA and isopropanol were mixed and therefore no products could be recovered.

September 20, 2010

ADS

<b>Objective:</b> Analyze PCR of mms6 (HB) and fluorescent proteins (AV).
<b>Method:</b> Analyzed on 2% TAE agarose gel.
Load order:

Lane	Sample	Volume Sample (µL)	Volume Loading Dye (µL)
1	HB1	10	2
2	HB2	10	2
3	HB3	10	2
4	HB4	10	2
5	HB5	10	2
6	100bp Ladder (Fermentas)	0.5	2(+10 H20)
7	RFP1	10	2
8	RFP2	10	2
9	RFP3	10	2

Results: ADD IMAGE

• mms6 was not amplified
• RFP was amplified
  • Expected size is ~800bp
  • Actual size is >1000bp
    • We believe that the suffix segment of the fusion primer annealed rather than the FP segment. This would cause the promoter (R0040) of J04450 to be amplified, adding ~200bp, giving a final size of >1000bp.

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Objective: Confirm assembly (3 antibiotic) of K118021 and B0015 from Sept. XX, 2010 via PCR.
Method: Set up 50uL reaction mixture with 2uL of ligated DNA. Used VF2 and VR primers.
Used PFU setting on Thermocycler
Results: ADD IMAGE
Conclusion: Assembly did not work as intended.

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HB

Objective: PCR mms6 to attach fusion standard to the N-term and standard suffix to C-term.

Component	1X(µL)	Master Mix(x6)(µL)
Milli-Q H2O	12.8	76.8
10x Pfu Buffer with MgSO4	2	12
dNTPs	1	6
Fusion Forward Primer	1	6
Standard Reverse Primer	1	6
Template DNA	2	12
Pfu polymerase	0.2	1.2

Prefix fusion sense primer has melting temperature of 56.1^oC. Standard suffix primer has melting temperature of 61.3^oC.

PCR- Conditions: 1. 95^oC for 180 sec 2. 95^oC for 30 sec 3. 51.1^oC for 30 sec 4. 72^oC for 150 sec 5. 72^oC for 600 sec (35 cycles)

Program HBPREFU: 1. 49.1^oC 2. 50.2^oC 3. 51.4^oC 4. 52.6^oC 5. 53.5^oC

September 21, 2010

ADS

Objective: Insert xylE (with N and C terminal fusion standards, obtained by PCR of K118021 by KG) into pSB1C3 plasmid for submission to registry.

Method:

• Restriction of xylE PCR product and pSB1C3 (containing J04450 biobrick) via BioBrick Method using EcoRI-HF and PstI (Enzymes from NEB)
• Ligation of xylE PCR product and pSB1C3 via BioBrick Method using T4 DNA ligase (Enzyme from NEB)
• Transform into Library Efficiency Compentent DH5α Cells (Invitrogen)

Results:

• Obtained too numerous to count (TNTC) colonies.
  • Obtained ~100 white colonies (indicating removal of RFP and insertion of new BioBrick)

Note:
Parent plasmid (from PCR) not digested, possibly K118021 moved into pSB1C3 backbone.

Follow-up:
Screen white colonies by addition of catechol to solution containing white cells.

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TF, MC

Objective: Obtain a preparation of B0015 (dT).

Method: Used Plasmid DNA Purification by Alkaline Lysis (Large Scale AKA Maxiprep protocol.

September 21, 2010

ADS

<b>Objective:</b> Analyze PCR of K118021-B0015 and subsequent PCR (ADS) and PCR of <partinfo>K118021</partinfo> to add either N or C terminal fusion standard (KG).
<b>Method:</b> Analyze on 2% TAE agarose gel.
Load Order:

Lane	Sample	Volume Sample (µL)	Volume Loading Dye (µL)
1	xylE F-S 1	5	1
2	xylE F-S 2	5	1
3	xylE F-S 3	5	1
4	xylE F-S 4	5	1
5	xylE F-S 5	5	1
6	xylE F-S 6	5	1
7	100bp Ladder (NEB)	0.5	1(+5 H20)
8	xylE S-F 1	5	1
9	xylE S-F 2	5	1
10	xylE S-F 3	5	1
11	xylE S-F 4	5	1
12	xylE S-F 5	5	1
13	xylE S-F 6	5	1
14	Empty
15	PCR of K118021-B0015 (ADS)	1	1
16	Empty
17	Empty

Results: ADD IMAGE

• Both KG PCR (Standard-Fusion and Fusion-Standard) amplified
• ADS PCR Amplified, but no insert present.

September 23, 2010

JV

Objective: Characterized catechol degradation by xylE enzyme

Method: Measured absorbance of catechol (275nm) and 2-hydroxymuconate semialdehyde (380nm).

• Protocol:
• 1) Grow cells in M9 minimal medium
• 2) Take 1/10 dilution of cells
• 3) Introduce 1µL of 0.05M catechol solution into the cell dilution. (Final concentration of 50&microM;).
• 4) Quench the reaction with 5A% w/v trichloroacetate at certain time points. (0,15sec, 30sec, 45sec, 60sec, 2min, 3min, 4min, 5min, 10min).
• 5) Spin down cells.
• 6) Measure absorbance of supernatant.
  

Results: Cuvette used interfered with Spectra.

ADS

NOTE: In all transformations, heat shock step was missed. HOWEVER, all transformations showed significant number of colony forming units.
Objective: Move xylE (two biobrick; one with Fusion prefix, one with fusion suffix) into pSB1C3.
Method:

• Restriction of xylE PCR product and pSB1C3 (containing J04450 biobrick) via BioBrick Method using EcoRI-HF and PstI (Enzymes from NEB)
• Ligation of xylE PCR product and pSB1C3 via BioBrick Method using T4 DNA ligase (Enzyme from NEB)
  • Incubated 30 min at RT
• Transform into Subcloning Efficiency Compentent DH5α Cells (Invitrogen)

Results: TBD
Follow-up: TBD

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Objective: Create glycerol stocks of <partinfo>J04450</partinfo> in pSB1A3 and pSB1T3 for use in RFP-BioBrick Assembly.
Method: Transform into Subcloning Efficiency Competent DH5α Cells (Invitrogen)
Obtained all plasmid DNA from 2010 Kit Plate 1

• J04450 in pSB1A3 - Well 1C
• J04450 in pSB1T3 - Well 7A

Results: Obtained TNTC colonies
Follow-up:

• Grow overnight cultures
• Generate Glycerol Stocks
• Generate Plasmid DNA via Maxiprep

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Objective: Create glycerol stocks of received synthesized (Mr. Gene) signal peptides.
Method: Transform into Subcloning Efficiency Competent DH5α Cells (Invitrogen) plasmid DNA containing the following BioBricks:

• 1) <partinfo>K331007</partinfo> - β-lactamase Bla Signal Sequence
• 2) <partinfo>K331008</partinfo> - Outer Membrane Protein ompA
• 3) <partinfo>K331009</partinfo> - Heat Stable Toxin I
• 4) <partinfo>K331012</partinfo> - Penicillin Binding Protein DacA
• All inserts in pMA-T vector (Standard Mr. Gene vector)

Results: Obtained TNTC Cells
Follow-up:

• 1) Grow overnight cultures
• 2) Purify pDNA
• 3) Move into pSB1C3 plasmid
• 4) Verify sequence
• 5) Submit to registry for sequencing

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TF, MC

Objective: Obtain a preparation of J04450 (RFP).

Method: Used Plasmid DNA Purification by Alkaline Lysis (Large Scale AKA Maxiprep protocol.

September 24, 2010

ADS

<b>Objective:</b> Generate plasmid DNA of <partinfo>E1010</partinfo> for downstream PCR
<b>Method:</b> Transform plasmid DNA into Subcloning Efficiency Competent DH5α Cells (Invitrogen)
DNA obtained from 2010 Kit Plate 1 Well 18F (E1010 in pSB2K3)
<b>Results:</b> Obtained TBD colonies
<b>Follow-up:</b>

• Grow overnight cultures (Generate glycerol stocks)
• Purify plasmid DNA (Generate pDNA stocks)
• PCR to add terminal fusion standards

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<b>Objective:</b> Create glycerol stocks of J04450 in pSB1K3 for use in RFP-BioBrick Assembly.
<b>Method:</b> Transform into Subcloning Efficiency Competent DH5α Cells (Invitrogen)
Obtained plasmid DNA from 2010 Kit Plate 1 well 5A (J04450 in pSB1K3)
<b>Results:</b> Obtained TNTC colonies
<b>Follow-up:</b>

• Grow overnight cultures
• Generate Glycerol Stocks
• Generate Plasmid DNA via Maxiprep

September 25, 2010

JV

<b>Objective:</b> Extract Plasmid DNA from DH5α cells.

<b>Method:</b>Qiagen spin column protocol.

• <partinfo>K331007</partinfo> (in pMA-T vector)
• <partinfo>K331008</partinfo> (in pMA-T vector)
• <partinfo>K331009</partinfo> (in pMA-T vector)
• <partinfo>K331012</partinfo> (in pMA-T vector)
  

Cells containing plasmids were put into glycerol stocks and put into HJ's -80^oC.

ADS

<b>Objective:</b> Move plasmid DNA made by JV (above, Sept 25, 2010) into pSB1C3 for submission to the Standard Registry of Parts.
<b>Method:</b>

• 1) Digest at 37^o for 10 min with PstI and EcoRI:
  • pSB1C3 containing <partinfo>J04450</partinfo>
  • pMA-T with <partinfo>K331007</partinfo>, <partinfo>K331008</partinfo>, <partinfo>K331009</partinfo>, and <partinfo>K331012</partinfo>.
• 2) Heat Kill at 80^oC for 20 min
• 3) Mix 2µL of pSB1C3 with each biobrick, ligate with T4 DNA ligase for 10 min
• 4) Transform into subcloning efficiency DH5α cells, plate on ampicillin plates.
  

<b>Results:</b> Obtained RESULT!!! positive colonies

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<b>Objective:</b> Assemble KG's C-terminal fusion xylE (<partinfo>K331021</partinfo>) (UNCONFIRMED) and C-terminal Arginine + dT (<partinfo>S04261</partinfo>) to create <partinfo>K331011</partinfo>.
<b>Method:</b>

• 1) Digest at 37^o for 10 min:
  • pSB1C3 containing <partinfo>J04450</partinfo> with PstI and EcoRI
  • <partinfo>K331021</partinfo> with EcoRI, SpeI
  • <partinfo>S04261</partinfo> with XbaI and PstI
• 2) Heat Kill at 80^oC for 20 min
• 3) Mix 2µL of pSB1C3 with each biobrick, ligate with T4 DNA ligase for 10 min
• 4) Transform into subcloning efficiency DH5α cells, plate on ampicillin plates.
  

<b>Results:</b> Obtained RESULT!!! positive colonies

September 26, 2010

ADS

<b>Objective:</b> Purify plasmid DNA from 5mL cultures grown from transformations of KG's ligated PCRs containing (UNCONFIRMED) <partinfo>K331020</partinfo> and <partinfo>K331021</partinfo>.
<b>Method:</b> Used Qiagen spin column method with BioBasic EZ-10 spin columns.

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<b>Objective:</b> Assemble the following:

• <partinfo>K331020</partinfo> + <partinfo>B0015</partinfo> to obtain To be named biobrick
• <partinfo>K331021</partinfo> + <partinfo>S04261</partinfo> to obtain To be named biobrick

<b>Method:</b>

• Digest <partinfo>K331020</partinfo> and <partinfo>K331021</partinfo> with EcoRI and SpeI
• Digest <partinfo>B0015</partinfo> and <partinfo>S04261</partinfo> with XbaI and PstI
• Digest <partinfo>J04450</partinfo> in pSB1C3 with EcoRI and PstI

Incubate each at 37^oC for 10 min
Heat kill at 80^oC for 20 min
Ligate with T4 Ligase for 10 min at room temperature
Transform into DH5&alpha subcloning efficiency cells
<b>Results:</b> Obtained RESULTS!!!! positive colonies.
Also analyzed minipreps on 1.5% TAE Agarose Gel
GEL!!!

September 29, 2010

ADS

Received synthesized parts from Mr. GENE (all in pMA vectors)

• <partinfo>K331022</partinfo>
• <partinfo>K331023</partinfo>
• <partinfo>K331024</partinfo>
• <partinfo>K331025</partinfo>

Each tube contained 5µg pDNA; reconstitute in 50µL of TE buffer to get 100ng/µL concentration
Transform 1µL into DH5α subcloning efficiency cells
Obtained TNTC colonies
Started overnight 5mL cultures for miniprep and insertion into pSB1C3 vector.

Retrieved from "http://2010.igem.org/Team:Lethbridge/Notebook/Lab_Work/September"