{{HarvardFancybox}}

{{HarvardFancybox}}

{{harvard fence}}

{{harvard fence}}

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__NOTOC__

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| Week 1  || [[#06-14-2010|6-14-2010]] || [[#06-15-2010|06-15-2010]] || [[#06-16-2010|06-16-2010]] || [[#06-17-2010|06-17-2010]] || [[#06-18-2010|06-18-2010]]

| Week 1  || [[#06-14-2010|6-14-2010]] || [[#06-15-2010|06-15-2010]] || [[#06-16-2010|06-16-2010]] || [[#06-17-2010|06-17-2010]] || [[#06-18-2010|06-18-2010]]

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| Week 3  || [[#06-28-2010|06-28-2010]] || [[#06-29-2010|06-29-2010]] || [[#06-30-2010|06-30-2010]] || [[#07-01-2010|07-01-2010]] || [[#07-02-2010|07-02-2010]]

| Week 3  || [[#06-28-2010|06-28-2010]] || [[#06-29-2010|06-29-2010]] || [[#06-30-2010|06-30-2010]] || [[#07-01-2010|07-01-2010]] || [[#07-02-2010|07-02-2010]]

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| Week 5  || [[#07-12-2010|07-12-2010]] || [[#07-13-2010|07-13-2010]] || [[#07-14-2010|07-14-2010]] || [[#07-15-2010|07-15-2010]] || [[#07-16-2010|07-16-2010]]

| Week 5  || [[#07-12-2010|07-12-2010]] || [[#07-13-2010|07-13-2010]] || [[#07-14-2010|07-14-2010]] || [[#07-15-2010|07-15-2010]] || [[#07-16-2010|07-16-2010]]

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|-

|-

|-

|-

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| Week 8  || [[#08-02-2010|08-02-2010]] || [[#08-03-2010|08-03-2010]] || [[#08-04-2010|08-04-2010]] || [[#08-05-2010|08-05-2010]] || [[#08-06-2010|08-06-2010]]

| Week 8  || [[#08-02-2010|08-02-2010]] || [[#08-03-2010|08-03-2010]] || [[#08-04-2010|08-04-2010]] || [[#08-05-2010|08-05-2010]] || [[#08-06-2010|08-06-2010]]

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<b>LacI Transformation</b>

<b>LacI Transformation</b>

Transformed the following, each into its own tube of TOP10 E.Coli:

Transformed the following, each into its own tube of TOP10 E.Coli:

Plates incubated overnight, colonies observed in all plates except the control.

Plates incubated overnight, colonies observed in all plates except the control.

the following sequences for LacI and a Nuclear Localization Signal (NLS) were ordered for PCR:

the following sequences for LacI and a Nuclear Localization Signal (NLS) were ordered for PCR:

     5'-CTA GAT CTT CTG TTG TTC ATC CTA AGA AGA AGA GAA AGG TTT GAA CTA GTA GCG GCC GCT GCA -3'

     5'-CTA GAT CTT CTG TTG TTC ATC CTA AGA AGA AGA GAA AGG TTT GAA CTA GTA GCG GCC GCT GCA -3'

Performed bacterial transformation according to [[Silver:_Bacterial_Transformation]], however we used 1μL in step 3, and used entire tube of TOP10 competent cells instead of only 10-15μL.

Performed bacterial transformation according to [[Silver:_Bacterial_Transformation]], however we used 1μL in step 3, and used entire tube of TOP10 competent cells instead of only 10-15μL.

Prepared 6 overnight cell cultures, consisting of three colonies each from the LacI wt and LacI+rapid degradation tail E.Coli cells transformed previously.

Prepared 6 overnight cell cultures, consisting of three colonies each from the LacI wt and LacI+rapid degradation tail E.Coli cells transformed previously.

Performed miniprep of overnight cell cultures of 3 colonies each of wildtype LacI transformed bacteria and LacI+rapid degradation tail bacteria.

Performed miniprep of overnight cell cultures of 3 colonies each of wildtype LacI transformed bacteria and LacI+rapid degradation tail bacteria.

Minipreps performed according to pages 22-23 of the [http://www.google.com/url?sa=t&source=web&cd=1&ved=0CBIQFjAA&url=http%3A%2F%2Fkirschner.med.harvard.edu%2Ffiles%2Fprotocols%2FQIAGEN_QIAprepMiniprepKit_EN.pdf&ei=wOcbTOfxCIH-8AaIhI2ADA&usg=AFQjCNENLjIQI2lUNMlpIqkcKrrq9buqLg&sig2=GVeWspXGiA0bwGnulA18-Q QIAprep Miniprep Handbook].

Minipreps performed according to pages 22-23 of the [http://www.google.com/url?sa=t&source=web&cd=1&ved=0CBIQFjAA&url=http%3A%2F%2Fkirschner.med.harvard.edu%2Ffiles%2Fprotocols%2FQIAGEN_QIAprepMiniprepKit_EN.pdf&ei=wOcbTOfxCIH-8AaIhI2ADA&usg=AFQjCNENLjIQI2lUNMlpIqkcKrrq9buqLg&sig2=GVeWspXGiA0bwGnulA18-Q QIAprep Miniprep Handbook].

Blanked nanodrop sensor with 2μL EB buffer, then measured each of the 6 minipreped LacI plasmids.

Blanked nanodrop sensor with 2μL EB buffer, then measured each of the 6 minipreped LacI plasmids.

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We digested 15μL of each of the 6 samples with 1μLH₂O, 1μL EcoR1, and 1μL Pst1 (high efficiency restriction enzymes).

We digested 15μL of each of the 6 samples with 1μLH₂O, 1μL EcoR1, and 1μL Pst1 (high efficiency restriction enzymes).

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Prepared 3 10cc tubes each containing .5μL LB+amp and cells from one of the three colonies of the VP16 cell culture of the previous night (colonies from the second-tube plate, the cells in which were heatshocked in the correct order).

Prepared 3 10cc tubes each containing .5μL LB+amp and cells from one of the three colonies of the VP16 cell culture of the previous night (colonies from the second-tube plate, the cells in which were heatshocked in the correct order).

Tubes were set to shake at 37°C overnight.

Tubes were set to shake at 37°C overnight.

Prepared 2 cryo tubes, with the following labeling:

Prepared 2 cryo tubes, with the following labeling:

Both Cryo tubes were placed in the box labeled 'Genetic Fence iGEM 2010' in the -80°C freezer.

Both Cryo tubes were placed in the box labeled 'Genetic Fence iGEM 2010' in the -80°C freezer.

Following QIAgen Plasmid DNA Purification Protocol Using the QIAprep Spin Miniprep Kit and a Microcentrifuge the following plasmid was purified:

Following QIAgen Plasmid DNA Purification Protocol Using the QIAprep Spin Miniprep Kit and a Microcentrifuge the following plasmid was purified:

*VP16<br>

*VP16<br>

*4ml from each overnight cell culture of VP16 (1,2&3) were transferred into 5 ml conicals and spun for 6 minutes at 4400 rpm<br>

*4ml from each overnight cell culture of VP16 (1,2&3) were transferred into 5 ml conicals and spun for 6 minutes at 4400 rpm<br>

*flow through kept and purity tested<br>

*flow through kept and purity tested<br>

*VP16 #1

*VP16 #1

**260/280 = 1.92

**260/280 = 1.92

Quantities per double digestion reaction

Quantities per double digestion reaction

*1 unit enzyme cuts 1 μg DNA in 30-60 minutes

*1 unit enzyme cuts 1 μg DNA in 30-60 minutes

Ingredients

Ingredients

BBa_K105007, plate 3 well 9I, psB1A2

BBa_K105007, plate 3 well 9I, psB1A2

*streaked on LB + amp paltes, incubated at 37°C overnight

*streaked on LB + amp paltes, incubated at 37°C overnight

LacI+NLS primers arrived in mail

LacI+NLS primers arrived in mail

used LACIN program in phusion PCR machine to perform PCR

used LACIN program in phusion PCR machine to perform PCR

Barnase and Barstar Plasmids from ADDGENE arrived in mail

Barnase and Barstar Plasmids from ADDGENE arrived in mail

Karmella suggested we try adding an LVA tail to the Cre we use for the crelox fence system.

Karmella suggested we try adding an LVA tail to the Cre we use for the crelox fence system.

Pipetted 5mL of LB+Amp each into capped cc tubes, and mixed bacterial scrapings from 5 separate colonies of transformed GAL4 DNA binding domain E.Coli. Set to shake at 37 degrees C overnight.

Pipetted 5mL of LB+Amp each into capped cc tubes, and mixed bacterial scrapings from 5 separate colonies of transformed GAL4 DNA binding domain E.Coli. Set to shake at 37 degrees C overnight.

*pipetted 4 ml from each overnight cell culture from colonies 1-5 into a 15 ml conical centrifuged at 4400 rpm for 6 minute

*pipetted 4 ml from each overnight cell culture from colonies 1-5 into a 15 ml conical centrifuged at 4400 rpm for 6 minute

*tubes labeled "GAL4" plus the specific colony number

*tubes labeled "GAL4" plus the specific colony number

PCR program for LacIN modified after Monday's unsuccessful O2 and E10 PCR (no product)

PCR program for LacIN modified after Monday's unsuccessful O2 and E10 PCR (no product)

7=4°C for ∞

7=4°C for ∞

PCR for E10 LacI

PCR for E10 LacI

*The above settings on the PCR machine were used

*The above settings on the PCR machine were used

To Make Agarose Gel

To Make Agarose Gel

*150 mL TAE buffer combined with 1.5 g agarose in beaker

*150 mL TAE buffer combined with 1.5 g agarose in beaker

see [http://www.qiagen.com/products/dnacleanup/gelpcrsicleanupsystems/qiaquickgelextractionkit.aspx#Tabs=t2 QIAquick spin handbook]

see [http://www.qiagen.com/products/dnacleanup/gelpcrsicleanupsystems/qiaquickgelextractionkit.aspx#Tabs=t2 QIAquick spin handbook]

Colonies from the following cultures were innoculated and set to shake at 37°C overnight:

Colonies from the following cultures were innoculated and set to shake at 37°C overnight:

*lox71

*lox71

The following colonies were minipreped after passing the night in the shaking incubator (37°C)

The following colonies were minipreped after passing the night in the shaking incubator (37°C)

* PMT413

* PMT413

* lox71

* lox71

Blanked nanodrop sensor with 2μL EB buffer, then measured each of the 6 minipreped LacI plasmids.

Blanked nanodrop sensor with 2μL EB buffer, then measured each of the 6 minipreped LacI plasmids.

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Primers:

Primers:

Our first digestion was unsuccessful except for the confirmation of the PMT316 fragment; this can be attributed to an incorrect combination of restriction enzymes.  The digestion was performed again in the afternoon using EcoRI + SpeI for the biobricked plasmids (lox71, GFP mut, lox66, cre recombinase, and firefly luciferase) and EcoRI + HindIII for PMT413.

Our first digestion was unsuccessful except for the confirmation of the PMT316 fragment; this can be attributed to an incorrect combination of restriction enzymes.  The digestion was performed again in the afternoon using EcoRI + SpeI for the biobricked plasmids (lox71, GFP mut, lox66, cre recombinase, and firefly luciferase) and EcoRI + HindIII for PMT413.

Gel Image, first digestion:

Gel Image, first digestion:

PMT316 digests in lanes 8 and 9 show bands of approximately 439 bp. (above image)

PMT316 digests in lanes 8 and 9 show bands of approximately 439 bp. (above image)

The length of the shortest bands were confirmed to be roughly the same length of the desired excerpt with the exception of Lox66 and Lox71 where it was determined that the inserts being only 34 bp had run off the gel, therefore the single band seen was the remaining plasmid.

The length of the shortest bands were confirmed to be roughly the same length of the desired excerpt with the exception of Lox66 and Lox71 where it was determined that the inserts being only 34 bp had run off the gel, therefore the single band seen was the remaining plasmid.

To assay the effect of Methoxyfenozide on yeast, three flasks containing 25μL DMSO (control), 2.5μL of 100mM Methoxyfenozide stock in 25mL DMSO to form the 1μM condition, and 25μL of 100mM Methoxyfenozide stock in 25mL DMSO to from the 10μM condition.

To assay the effect of Methoxyfenozide on yeast, three flasks containing 25μL DMSO (control), 2.5μL of 100mM Methoxyfenozide stock in 25mL DMSO to form the 1μM condition, and 25μL of 100mM Methoxyfenozide stock in 25mL DMSO to from the 10μM condition.

Each beaker also contained y190 yeast strains and YPD medium.

Each beaker also contained y190 yeast strains and YPD medium.

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Inoculated 3 colonies of B21 transformed E.Coli and set them to shake for 8 hours. We will maxiprep the B21 plasmid for use as a vector backbone.

Inoculated 3 colonies of B21 transformed E.Coli and set them to shake for 8 hours. We will maxiprep the B21 plasmid for use as a vector backbone.

Ran 3 tubes of each for a total of 6.

Ran 3 tubes of each for a total of 6.

7x Mastermix:

7x Mastermix:

Reduced annealing temp to 50°C from 56°C which was the last attempt, ran otherwise identical LACIN program

Reduced annealing temp to 50°C from 56°C which was the last attempt, ran otherwise identical LACIN program

Ran 1.2% E-gel of the 6 PCR product tubes.

Ran 1.2% E-gel of the 6 PCR product tubes.

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Because colonies 1 and 2 of Barstar worked properly on last week's gel, the PCR products in those two tubes were combined and purified using the Qiagen PCR purification protocol.

Because colonies 1 and 2 of Barstar worked properly on last week's gel, the PCR products in those two tubes were combined and purified using the Qiagen PCR purification protocol.

*as there was about 25μL of PCR product in the two together, 125μL of PB buffer was added (5x the concentration of the PCR product)

*as there was about 25μL of PCR product in the two together, 125μL of PB buffer was added (5x the concentration of the PCR product)

*labeled the eppendorf tube 'Barstar PCR purified'

*labeled the eppendorf tube 'Barstar PCR purified'

B21 plasmid maxiprep

B21 plasmid maxiprep

followed instructions as described in the QIAgen Plasmid Purification Handbook pg. 19-23. (2005 edition)

followed instructions as described in the QIAgen Plasmid Purification Handbook pg. 19-23. (2005 edition)

*4ml from each overnight cell culture of pMT1002 (1,2&3) were transferred into 5 ml conicals and spun for 6 minutes at 4400 rpm<br>

*4ml from each overnight cell culture of pMT1002 (1,2&3) were transferred into 5 ml conicals and spun for 6 minutes at 4400 rpm<br>

Also see pages 22-23 of the [http://www.google.com/url?sa=t&source=web&cd=1&ved=0CBIQFjAA&url=http%3A%2F%2Fkirschner.med.harvard.edu%2Ffiles%2Fprotocols%2FQIAGEN_QIAprepMiniprepKit_EN.pdf&ei=wOcbTOfxCIH-8AaIhI2ADA&usg=AFQjCNENLjIQI2lUNMlpIqkcKrrq9buqLg&sig2=GVeWspXGiA0bwGnulA18-Q QIAprep Miniprep Handbook] for miniprep instructions.

Also see pages 22-23 of the [http://www.google.com/url?sa=t&source=web&cd=1&ved=0CBIQFjAA&url=http%3A%2F%2Fkirschner.med.harvard.edu%2Ffiles%2Fprotocols%2FQIAGEN_QIAprepMiniprepKit_EN.pdf&ei=wOcbTOfxCIH-8AaIhI2ADA&usg=AFQjCNENLjIQI2lUNMlpIqkcKrrq9buqLg&sig2=GVeWspXGiA0bwGnulA18-Q QIAprep Miniprep Handbook] for miniprep instructions.

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20μL per tube

20μL per tube

put 10x green FD buffer on ice to thaw.

put 10x green FD buffer on ice to thaw.

*14.3μL H₂O (to bring the total to 20μL)

*14.3μL H₂O (to bring the total to 20μL)

Pst1 and Xba1 digest of B21 plasmid, lane 4 and 5 cut, 7 and 8 uncut

Pst1 and Xba1 digest of B21 plasmid, lane 4 and 5 cut, 7 and 8 uncut

Second Attempt

Second Attempt

Lane 2 buffer, lane 8 and 9 Colony B, lanes 15 and 16 colony A

Lane 2 buffer, lane 8 and 9 Colony B, lanes 15 and 16 colony A

Cut out the backbone (upper band, and placed into four eppendorf tubes, labeled 'B21 backbone' and with their respective weight before the gel was added. Placed all four in the iGEM team fence box in the -4°C freezer to be purified tomorrow.

Cut out the backbone (upper band, and placed into four eppendorf tubes, labeled 'B21 backbone' and with their respective weight before the gel was added. Placed all four in the iGEM team fence box in the -4°C freezer to be purified tomorrow.

*Designed primer sequences for ACC synthase degradation site, linker sequence connecting Barnase to the degradation site.

*Designed primer sequences for ACC synthase degradation site, linker sequence connecting Barnase to the degradation site.

*Designed primers to amplify germination/embryogenesis promoter and AtArp2 promoter.

*Designed primers to amplify germination/embryogenesis promoter and AtArp2 promoter.

B21 Backbone Gel Extraction

B21 Backbone Gel Extraction

*followed QIAquick gel extraction handbook except melting temperature was 42°C instead of 50°C

*followed QIAquick gel extraction handbook except melting temperature was 42°C instead of 50°C

only one band was visible in the gel for each reaction.  Julia forgot to put the ladder in so it was not possible to precisely determine the single band size, but judging from the position of the loading dye it seemed that the plasmid had only been cut once.

only one band was visible in the gel for each reaction.  Julia forgot to put the ladder in so it was not possible to precisely determine the single band size, but judging from the position of the loading dye it seemed that the plasmid had only been cut once.

Ligation performed on Barstar, NLS, and LacIN

Ligation performed on Barstar, NLS, and LacIN

*incubate at 37°C overnight

*incubate at 37°C overnight

Digested with EcoR1 and HindIII

Digested with EcoR1 and HindIII

Lane 6 pMT413

Lane 6 pMT413

Standard PCR measurements were used except that a new 50X dNTP mix was used in place of the 5X buffer used in all previous PCR reactions.  The amount of dH20 was adjusted for the new quantity of dNTP mix accordingly.

Standard PCR measurements were used except that a new 50X dNTP mix was used in place of the 5X buffer used in all previous PCR reactions.  The amount of dH20 was adjusted for the new quantity of dNTP mix accordingly.

Inoculated 7 Barstar and 3 NLS colonies from Thursday's ligation.

Inoculated 7 Barstar and 3 NLS colonies from Thursday's ligation.

*31μL H2O

*31μL H2O

Digestion successful, larger bands removed for gel extraction.

Digestion successful, larger bands removed for gel extraction.

Gel extraction performed according to QIAgen gel extraction protocol.

Gel extraction performed according to QIAgen gel extraction protocol.

Performed miniprep of overnight cell cultures of 7 colonies of Barstar, 3 colonies of NLS.

Performed miniprep of overnight cell cultures of 7 colonies of Barstar, 3 colonies of NLS.

Minipreps performed according to pages 22-23 of the [http://www.google.com/url?sa=t&source=web&cd=1&ved=0CBIQFjAA&url=http%3A%2F%2Fkirschner.med.harvard.edu%2Ffiles%2Fprotocols%2FQIAGEN_QIAprepMiniprepKit_EN.pdf&ei=wOcbTOfxCIH-8AaIhI2ADA&usg=AFQjCNENLjIQI2lUNMlpIqkcKrrq9buqLg&sig2=GVeWspXGiA0bwGnulA18-Q QIAprep Miniprep Handbook].

Minipreps performed according to pages 22-23 of the [http://www.google.com/url?sa=t&source=web&cd=1&ved=0CBIQFjAA&url=http%3A%2F%2Fkirschner.med.harvard.edu%2Ffiles%2Fprotocols%2FQIAGEN_QIAprepMiniprepKit_EN.pdf&ei=wOcbTOfxCIH-8AaIhI2ADA&usg=AFQjCNENLjIQI2lUNMlpIqkcKrrq9buqLg&sig2=GVeWspXGiA0bwGnulA18-Q QIAprep Miniprep Handbook].

7 reactions of Barstar and 3 reactions of NLS

7 reactions of Barstar and 3 reactions of NLS

3 reactions, each with:

3 reactions, each with:

*1μL Fwd LacIN Primer

*1μL Fwd LacIN Primer

5 reactions each of Barnase from pMT413, Barnase from pMT1002, and Gal4

5 reactions each of Barnase from pMT413, Barnase from pMT1002, and Gal4

*10.5μL DH₂O

*10.5μL DH₂O

After receiving the new primers we performed PCR on barnase vectors PMT413 and PMT1002, LacIN, as well as the GAL4 DNA binding domain.  The ladder is in lane 2, the GAL4 PCR products are in lanes 5 through 9, the barnase PMT413 lanes 10 through 14, and the LacIN PCR product is in the last 3 lanes.  After PCR was confirmed successful, we decided to gel purify the LacIN because of the multiple band sizes seen in the gel.  Below is the gel where LacIN was gel extracted, the chunk missing from the gel is where LacIN was identified to be.

After receiving the new primers we performed PCR on barnase vectors PMT413 and PMT1002, LacIN, as well as the GAL4 DNA binding domain.  The ladder is in lane 2, the GAL4 PCR products are in lanes 5 through 9, the barnase PMT413 lanes 10 through 14, and the LacIN PCR product is in the last 3 lanes.  After PCR was confirmed successful, we decided to gel purify the LacIN because of the multiple band sizes seen in the gel.  Below is the gel where LacIN was gel extracted, the chunk missing from the gel is where LacIN was identified to be.

Gel extraction was performed according to the QIAgen gel extraction kit protocol using a microcentrifuge.

Gel extraction was performed according to the QIAgen gel extraction kit protocol using a microcentrifuge.

Submited plasmids from each of the 7 Barstar colonies for sequencing.

Submited plasmids from each of the 7 Barstar colonies for sequencing.

LacIN - 65μL

LacIN - 65μL

   1μL Xba1  

   1μL Xba1  

Eppendorfs labeled "LacIN digest cleanup 7/9," "Gal4 digest cleanup 7/9," and "Barnase digest cleanup 7/9"

Eppendorfs labeled "LacIN digest cleanup 7/9," "Gal4 digest cleanup 7/9," and "Barnase digest cleanup 7/9"

*Barnase, Gal4, LacIN

*Barnase, Gal4, LacIN

*4 cc tubes each, 5mL LB+amp, set to shake at 37°C overnight

*4 cc tubes each, 5mL LB+amp, set to shake at 37°C overnight

* No cells found in Barnase colonies 1 and 2 --> innoculations 3 and 4 renamed Barnase 1 and 2, replacing the cell-free cultures.

* No cells found in Barnase colonies 1 and 2 --> innoculations 3 and 4 renamed Barnase 1 and 2, replacing the cell-free cultures.

*centrifuged for 1 minute at 13,000 rpm

*centrifuged for 1 minute at 13,000 rpm

{| border="3" style="margin-left: 3em;"

{| border="3" style="margin-left: 3em;"

Xba1 and Pst1

Xba1 and Pst1

Gel of Digestions:

Gel of Digestions:

*submitted Barnase 1 and 2, Gal4 1,2, and 3, and LacIN 1,2,3, and 4 for sequencing

*submitted Barnase 1 and 2, Gal4 1,2, and 3, and LacIN 1,2,3, and 4 for sequencing

The yeast backbones containing the PIF3 and PHYB inserts was isolated and purified.  The latter image shows where the appropriately sized bands were excised.

The yeast backbones containing the PIF3 and PHYB inserts was isolated and purified.  The latter image shows where the appropriately sized bands were excised.

PIF3 20ng/μL

PIF3 20ng/μL

*resuspend DNA in approximately 200μL molecular grade pure water

*resuspend DNA in approximately 200μL molecular grade pure water

*add 1 volume phenol chloroform

*add 1 volume phenol chloroform

Ligation of NLS.Serine

Ligation of NLS.Serine

*2.5μL Backbone

*2.5μL Backbone

*1μL Quick Ligase

*1μL Quick Ligase

*1μL NLS lig into TOP10 cells

*1μL NLS lig into TOP10 cells

*Chilled on ice for 30 mins

*Chilled on ice for 30 mins

*Streaked on LB+amp plates, incubated overnight

*Streaked on LB+amp plates, incubated overnight

{| border="1"

{| border="1"

! 52(4)

! 52(4)

| 28.5 ||1.83

| 28.5 ||1.83

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Our previous attempt resulted in DNA with the following nanodrop values:

Our previous attempt resulted in DNA with the following nanodrop values:

*5 ng/μL 260/280: 1.48

*5 ng/μL 260/280: 1.48

PCR using the newly extracted DNA was unsuccessful.

PCR using the newly extracted DNA was unsuccessful.

The degradation signal from the Austrians was successfully inoculated and colonies were found on the agar plates.  Individual colonies from the plates were inoculated into tubes of 5 ml LB+Amp and placed in the shaking incubator overnight. (37°C)

The degradation signal from the Austrians was successfully inoculated and colonies were found on the agar plates.  Individual colonies from the plates were inoculated into tubes of 5 ml LB+Amp and placed in the shaking incubator overnight. (37°C)

*Made a 1 in 1000 dilution of the annealing reaction

*Made a 1 in 1000 dilution of the annealing reaction

Incubated at room temp for 15 mins, put on ice and immediately transformed.

Incubated at room temp for 15 mins, put on ice and immediately transformed.

*Thawed 3 Turbo chemically competent cells on ice

*Thawed 3 Turbo chemically competent cells on ice

*Pippeted 1μL of the ligation or annealing reaction into its own tube of turbo cells

*Pippeted 1μL of the ligation or annealing reaction into its own tube of turbo cells

*Streaked on LB + amp plates, and left in incubator overnight

*Streaked on LB + amp plates, and left in incubator overnight

Set ~400ml of water to boil in a beaker on a heat block.

Set ~400ml of water to boil in a beaker on a heat block.

*set in PCR machine at 16°C for 12 hours, then 4°C until stopped.

*set in PCR machine at 16°C for 12 hours, then 4°C until stopped.

*3μL NLS.Serine.Fwd

*3μL NLS.Serine.Fwd

*3μL NLS.Serine.Rev

*3μL NLS.Serine.Rev

Put in boiling water in beaker (see above), allowed to boil for 2 mins, then removed beaker from heat block, allowing the water to slowly cool with the annealing reaction tube still in it.

Put in boiling water in beaker (see above), allowed to boil for 2 mins, then removed beaker from heat block, allowing the water to slowly cool with the annealing reaction tube still in it.

Nanodrop of ligated annealed oligos:

Nanodrop of ligated annealed oligos:

5200.8 ng/μL,  1.32 260/280

5200.8 ng/μL,  1.32 260/280

poured 2% agarose Gel for gel extraction, ran 2μL of the ligated annealing reaction in 13μL EB buffer, with two KB plus 1 ladders, one on each side.

poured 2% agarose Gel for gel extraction, ran 2μL of the ligated annealing reaction in 13μL EB buffer, with two KB plus 1 ladders, one on each side.

Gel extraction performed, into an eppendorf weighing .9563g

Gel extraction performed, into an eppendorf weighing .9563g

Nanodrop for cleaned-up gel purified 5xGalpt negative - ran a 2% E-gel to check. There appears to be something at the .16kb size.

Nanodrop for cleaned-up gel purified 5xGalpt negative - ran a 2% E-gel to check. There appears to be something at the .16kb size.

Barstar samples 1, 2, 5, and 6 were confirmed to be correct.

Barstar samples 1, 2, 5, and 6 were confirmed to be correct.

Gal4 DBD sample 3 was unsuccessful.

Gal4 DBD sample 3 was unsuccessful.

FINALLY! EXP2 PROMOTER AMPLIFIED!

FINALLY! EXP2 PROMOTER AMPLIFIED!

~1000kb

~1000kb

Fragments were gel isolated and purified using the QIAgen Gel Purification Kit with the Gel Extraction protocol using a microcentrifuge.

Fragments were gel isolated and purified using the QIAgen Gel Purification Kit with the Gel Extraction protocol using a microcentrifuge.

No colonies from last night's ligations.

No colonies from last night's ligations.

Successful ACC synthase transformation:

Successful ACC synthase transformation:

PFX polymerase PCR

PFX polymerase PCR

total volume:100μL

total volume:100μL

*Thawed 2 turbo cells on ice to thaw

*Thawed 2 turbo cells on ice to thaw

*Resuspended EcR, RXRHm, RXRLc tubes from Mr.Gene into 50μL DH₂O (5μg into 50μL)

*Resuspended EcR, RXRHm, RXRLc tubes from Mr.Gene into 50μL DH₂O (5μg into 50μL)

*After 45 mins to 1.5 hours, streaked Act2LacOpt on Kan+LB plate, put in incubator to shake overnight.

*After 45 mins to 1.5 hours, streaked Act2LacOpt on Kan+LB plate, put in incubator to shake overnight.

*made 4 LB+Kanamycin plates

*made 4 LB+Kanamycin plates

   15μL Kanamycin

   15μL Kanamycin

Streaked this on LB agar plates with glass beads, and then turned the plate upside down and let dry until it was ready for bacterial sequencing, and then those same beads were reused.

Streaked this on LB agar plates with glass beads, and then turned the plate upside down and let dry until it was ready for bacterial sequencing, and then those same beads were reused.

Resuspended the following from the registry with 10μL DH₂O

Resuspended the following from the registry with 10μL DH₂O

   p(LAS)TetO, on pSB2K3 (kan res.), from plate 2, sell 11N

   p(LAS)TetO, on pSB2K3 (kan res.), from plate 2, sell 11N

   Terminator, on pSB1AK3 (kan and amp res.), from plate 1, well 4H

   Terminator, on pSB1AK3 (kan and amp res.), from plate 1, well 4H

*RXRHuman, RXRLocust, Barn1strep, Barn2strep, ACC, and EcR

*RXRHuman, RXRLocust, Barn1strep, Barn2strep, ACC, and EcR

(these are all amp resistant)

(these are all amp resistant)

The same is done in LB+Kan for pLAS and Act2lacO promoter, which are Kanamycin resistant.

The same is done in LB+Kan for pLAS and Act2lacO promoter, which are Kanamycin resistant.

poured a 2% agarose gel to gel-purify the barnase from  Barnase1 Xba Pst1 cleanup and Barnase2 Xba Pst1 cleanup, both from (7/23)

poured a 2% agarose gel to gel-purify the barnase from  Barnase1 Xba Pst1 cleanup and Barnase2 Xba Pst1 cleanup, both from (7/23)

*Gel ran empty except for DB ladders

*Gel ran empty except for DB ladders

*to prevent religation of the whole vector

*to prevent religation of the whole vector

*1μL Alkaline phosphatase

*1μL Alkaline phosphatase

Miniprepped and Nanodroped the following:

Miniprepped and Nanodroped the following:

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Again blank on the first attempt, tried again:

Again blank on the first attempt, tried again:

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