Team:Lethbridge/Notebook/Lab Work/September
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<th><a href="https://2010.igem.org/Team:Lethbridge/Notebook/Protocols"> | <th><a href="https://2010.igem.org/Team:Lethbridge/Notebook/Protocols"> | ||
<img src="https://static.igem.org/mediawiki/2010/9/91/UofLprotocolsbutton.jpg" width="60"/> | <img src="https://static.igem.org/mediawiki/2010/9/91/UofLprotocolsbutton.jpg" width="60"/> | ||
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</table><br> | </table><br> | ||
- | Added 48(µL) to each reaction. | + | Added 48(µL) to each reaction. <br> |
+ | [[image:Lethbridge_100903AssemblyADS.jpg|200px]] | ||
- | + | ---- | |
+ | <b>Objective:</b> Characterize xylE in LB Media vs. M9 Minimal Media.<br> | ||
+ | <b>Method:</b> Three experiments with spectra. <br> | ||
+ | |||
+ | * 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. | ||
==<font color="white">September 14, 2010== | ==<font color="white">September 14, 2010== | ||
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<b>Objective:</b> Miniprep of RFP expression construct (J04450) via Qiaquick/Qiaprep spin column. Protocol followed as per kit instructions.<br> | <b>Objective:</b> Miniprep of RFP expression construct (J04450) via Qiaquick/Qiaprep spin column. Protocol followed as per kit instructions.<br> | ||
- | ==<font color="white"> | + | ---- |
- | ===<font color="white"> | + | ===<font color="white">JV=== |
+ | |||
+ | <b>Objective:</b> PCR the pSB1C3 plasmid from part J04450 miniprep.<br> | ||
+ | <table border ="3"> | ||
+ | <tr><td><b>Component</b><td><b>1X(µL)</b> | ||
+ | <tr><td>Milli-Q H<sub>2</sub>O<td>12.8 | ||
+ | <tr><td>10x Pfu Buffer with MgSO<sub>4</sub><td>2 | ||
+ | <tr><td>dNTPs<td>1 | ||
+ | <tr><td>SB-prep-26 Primer<td>1 | ||
+ | <tr><td>SB-prep-3P Primer<td>1 | ||
+ | <tr><td>Template DNA<td>2 | ||
+ | <tr><td>Pfu polymerase<td>0.2 | ||
+ | </table><br> | ||
+ | |||
+ | Used iGEM cycle PSB1C3 in thermocycler. | ||
+ | Ran PCR product on 1% TAE agarose gel. | ||
+ | |||
+ | ---- | ||
+ | ===<font color="white">KG=== | ||
+ | |||
+ | <b>Objective:</b> PCR of xylE (mRBS-xylE K118021) with fusion standard so that an arginine tail can be attached.<br> | ||
+ | |||
+ | <table border ="3"> | ||
+ | <tr><td><b>Component</b><td><b>1X(µL)</b><td><b>Master Mix(x6)(µL)</b> | ||
+ | <tr><td>Milli-Q H<sub>2</sub>O<td>12.8<td>76.8 | ||
+ | <tr><td>10x Pfu Buffer with MgSO<sub>4</sub><td>2<td>12 | ||
+ | <tr><td>dNTPs<td>1<td>6 | ||
+ | <tr><td>xylE Fusion Prefix Forward Primer<td>1<td>6 | ||
+ | <tr><td>xylE Fusion Suffix Reverse Primer<td>1<td>6 | ||
+ | <tr><td>Template DNA<td>2<td>12 | ||
+ | <tr><td>Pfu polymerase<td>0.2<td>1.2 | ||
+ | </table><br> | ||
+ | |||
+ | PCR- Conditions: | ||
+ | 1. 95<sup>o</sup>C for 180 sec | ||
+ | 2. 95<sup>o</sup>C for 30 sec | ||
+ | 3. 56.6 +/- 5<sup>o</sup>C for 30 sec | ||
+ | 4. 72<sup>o</sup>C for 150 sec | ||
+ | 5. 72<sup>o</sup>C for 600 sec | ||
+ | (35 cycles) | ||
+ | |||
+ | Analyzed PCR on 2% agarose gel which ran at 100 V for 90 minutes.<br> | ||
+ | [[image:100915PCR AV KG JV.jpg|200px]] | ||
+ | |||
+ | ===<font color="white">AS=== | ||
<b>Objective:</b> Assemble xylE-dT using 3AB assembly and 3AB assembly.<br> | <b>Objective:</b> Assemble xylE-dT using 3AB assembly and 3AB assembly.<br> | ||
+ | |||
+ | Restriction Reactions - | ||
+ | |||
+ | 3AB Upstream Part (xylE) | ||
+ | <table><table border ="3"> | ||
+ | <tr><td><b>Ingredient</b></td><td>Volume(µL)</td></tr> | ||
+ | <tr><td>MilliQ H<sub>2</sub>0 Water</td><td>30.6</td></tr> | ||
+ | <tr><td>NEBuffer 2 (10x)</td><td>5</td></tr> | ||
+ | <tr><td>Plasmid DNA </td><td>11.9</td></tr> | ||
+ | <tr><td>EcoRI-HF</td><td>1</td></tr> | ||
+ | <tr><td>SpeI</td><td>1</td></tr> | ||
+ | <tr><td>100X BSA</td><td>0.5</td></tr> | ||
+ | </table> | ||
+ | |||
+ | 3AB Downstream Part (dT) | ||
+ | <table><table border ="3"> | ||
+ | <tr><td><b>Ingredient</b></td><td>Volume(µL)</td></tr> | ||
+ | <tr><td>MilliQ H<sub>2</sub>0 Water</td><td>24.6</td></tr> | ||
+ | <tr><td>NEBuffer 2 (10x)</td><td>5</td></tr> | ||
+ | <tr><td>Plasmid DNA</td><td>17.9</td></tr> | ||
+ | <tr><td>XbaI</td><td>1</td></tr> | ||
+ | <tr><td>PstI</td><td>1</td></tr> | ||
+ | <tr><td>100X BSA</td><td>0.5</td></tr> | ||
+ | </table> | ||
+ | |||
+ | 3AB Plasmid (pSB1C3) | ||
+ | <table><table border ="3"> | ||
+ | <tr><td><b>Ingredient</b></td><td>Volume(µL)</td></tr> | ||
+ | <tr><td>MilliQ H<sub>2</sub>0 Water</td><td></td></tr> | ||
+ | <tr><td>NEBuffer 2 (10x)</td><td>5</td></tr> | ||
+ | <tr><td>Plasmid DNA</td><td>42.5</td></tr> | ||
+ | <tr><td>EcoRI-HF</td><td>1</td></tr> | ||
+ | <tr><td>PstI</td><td>1</td></tr> | ||
+ | <tr><td>100X BSA</td><td>0.5</td></tr> | ||
+ | </table> | ||
+ | |||
+ | *Ran 10 minute and 60 minute reactions at 37<sup>o</sup>C. Followed by 20 minute heat shock at 80<sup>o</sup>C. | ||
+ | |||
+ | Ligation Reaction - | ||
+ | |||
+ | <table><table border ="3"> | ||
+ | <tr><td><b>Ingredient</b><td><b>1X(µL)</b> | ||
+ | <tr><td>MilliQ H<sub>2</sub>0 Water</td><td>11 | ||
+ | <tr><td>T4 Ligase Buffer (10x)</td><td>2 | ||
+ | <tr><td>Upstream Part</td><td>2 | ||
+ | <tr><td>Downstream Part </td><td>2 | ||
+ | <tr><td>T4 DNA Ligase</td><td>1 | ||
+ | <tr><td>Plasmid Part</td><td>2 | ||
+ | </table> | ||
+ | |||
+ | Incubated 10 minute and overnight ligations at room temperature ( 25<sup>o</sup>C). Heat killed ligase at 80<sup>o</sup>C 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<sup>o</sup>C 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<sup>o</sup>C | ||
+ | |||
+ | ==<font color="white">September 15, 2010== | ||
+ | ===<font color="white"> JV, ADS=== | ||
+ | <b>Objective:</b> Determine if dT ligated onto p-rbs-xylE using PCR analysis.<br> | ||
+ | |||
+ | <table border ="3"> | ||
+ | <tr><td><b>Component</b><td><b>1X(µL)</b><td><b>Master Mix(x4.5)(µL)</b> | ||
+ | <tr><td>Milli-Q H<sub>2</sub>O<td>11.8<td>53.1 | ||
+ | <tr><td>10x Pfu Buffer with MgSO<sub>4</sub><td>2<td>9 | ||
+ | <tr><td>dNTPs<td>1<td>4.5 | ||
+ | <tr><td>Forward VF2 Primer<td>1<td>4.5 | ||
+ | <tr><td>Reverse VR Primer<td>1<td>4.5 | ||
+ | <tr><td>Template DNA<td>1<td> | ||
+ | <tr><td>Pfu polymerase<td>0.2<td> | ||
+ | </table><br> | ||
+ | |||
+ | Used iGEM program 7 in thermocycler. | ||
+ | |||
+ | ---- | ||
+ | <b>Objective:</b> Determine if minipreps from Sept 15, 2010 worked. Analyze assembly intermediates. <br> | ||
+ | <b>Method:</b> Run 1% TAE agarose gels at 100 V for 90 minutes. <br> | ||
+ | |||
+ | [[image:100914ADSAssemblyPrep.jpg|100px]] | ||
+ | |||
+ | ==<font color="white">September 16, 2010== | ||
+ | ===<font color="white"> JV=== | ||
+ | <b>Objective:</b> Isolate RFP in pSB1C3 from DH5alpha cells.<br> | ||
+ | <b>Method:</b> Used Qiagen minipep protocol and spin column.<br> | ||
+ | <b>Results:</b> Gel of miniprepped RFP against previously prepared RFP showed a band of the right size.<br> | ||
+ | |||
+ | [[image:100916 JV RFP BW.jpg|50px]] | ||
+ | |||
+ | ==<font color="white">September 17, 2010== | ||
+ | ===<font color="white"> JV=== | ||
+ | <b>Objective:</b> Isolate plasmid DNA from DH5alpha cells: K249024; B0015; K118021/B0015<br> | ||
+ | <b>Method:</b> Used Qiagen minipep protocol and spin column. Ran a 1% agarose gel to view DNA<br> | ||
+ | |||
+ | GEL PICTURE! | ||
+ | ---- | ||
+ | ===<font color="white">AV=== | ||
+ | |||
+ | <b>Objective:</b> PCR out RFP from the complete construct (J04450) with a N-term fusion standard.<br> | ||
+ | |||
+ | Composition of each PCR tube: | ||
+ | <table><table border ="3"> | ||
+ | <tr><td><b>Component</b></td><td>Volume(µL)</td></tr> | ||
+ | <tr><td>10x Pfu Buffer w/ MgSO<sub>4</sub></td><td>2</td></tr> | ||
+ | <tr><td>dNTP (10mM)</td><td>1</td></tr> | ||
+ | <tr><td>N-term Fus Prefix</td><td>1</td></tr> | ||
+ | <tr><td>Biobrick Suffix</td><td>1</td></tr> | ||
+ | <tr><td>MilliQ H<sub>2</sub>O</td><td>12.8</td></tr> | ||
+ | <tr><td>Template DNA (J04450)</td><td>2</td></tr> | ||
+ | <tr><td>Pfu DNA Polymerase</td><td>0.2</td></tr> | ||
+ | </table> | ||
+ | |||
+ | PCR conditions: | ||
+ | <table><table border ="3"> | ||
+ | <tr><td><b>Step</b></td><td><b>Temperature (<sup>o</sup>C)</b></td><td><b>Time (mins)</b></td><td><b>Number of cycles</b></td></tr> | ||
+ | <tr><td>Initial Denaturation</td><td>95</td><td>2</td><td>1</td></tr> | ||
+ | <tr><td>Denaturation</td><td>95</td><td>0.5</td><td>25</td></tr> | ||
+ | <tr><td>Annealing</td><td>48.2, 52.4, 56.0 (gradient)</td><td>0.5</td><td>25</td></tr> | ||
+ | <tr><td>Extension</td><td>72</td><td>2</td><td>25</td></tr> | ||
+ | <tr><td>Final Extension</td><td>72</td><td>10</td><td>1</td></tr> | ||
+ | </table> | ||
+ | |||
+ | [[image:100915PCR AV KG JV.jpg|200px]] | ||
+ | |||
+ | Results: Amplification showing in gel, but fragment amplified was ~1000bp. RFP and dT should be ~800bp. Primers were checked and discovered the YFP/CFP primers were not compatible with the RFP. Concluded the amplification resulted from sloppy annealing of N-term fus prefix to the bio prefix resulting in the full construct being amplified (~1000bp) | ||
+ | ---- | ||
+ | |||
+ | ===<font color="white">KG=== | ||
+ | |||
+ | <b>Objective:</b> PCR xylE to attach fusion standard to the N-term and standard suffix to C-term.<br> | ||
+ | |||
+ | <table border ="3"> | ||
+ | <tr><td><b>Component</b><td><b>1X(µL)</b><td><b>Master Mix(x6)(µL)</b> | ||
+ | <tr><td>Milli-Q H<sub>2</sub>O<td>12.8<td>76.8 | ||
+ | <tr><td>10x Pfu Buffer with MgSO<sub>4</sub><td>2<td>12 | ||
+ | <tr><td>dNTPs<td>1<td>6 | ||
+ | <tr><td>Fusion Forward Primer<td>1<td>6 | ||
+ | <tr><td>Standard Reverse Primer<td>1<td>6 | ||
+ | <tr><td>Template DNA<td>2<td>12 | ||
+ | <tr><td>Pfu polymerase<td>0.2<td>1.2 | ||
+ | </table><br> | ||
+ | |||
+ | Fusion standard prefix has melting temperature of 66.5<sup>o</sup>C. Standard suffix has melting temperature of 70.5<sup>o</sup>C. | ||
+ | Annealing temperature of 61.5<sup>o</sup>C with 5 <sup>o</sup>C gradient. | ||
+ | PCR- Conditions: | ||
+ | 1. 95<sup>o</sup>C for 180 sec | ||
+ | 2. 95<sup>o</sup>C for 30 sec | ||
+ | 3. 61.5 +/- 5<sup>o</sup>C for 30 sec | ||
+ | 4. 72<sup>o</sup>C for 150 sec | ||
+ | 5. 72<sup>o</sup>C for 600 sec | ||
+ | (35 cycles) | ||
+ | |||
+ | ---- | ||
+ | <b>Objective:</b> PCR xylE to attach fusion standard to the C-term and standard suffix to N-term.<br> | ||
+ | |||
+ | <table border ="3"> | ||
+ | <tr><td><b>Component</b><td><b>1X(µL)</b><td><b>Master Mix(x6)(µL)</b> | ||
+ | <tr><td>Milli-Q H<sub>2</sub>O<td>12.8<td>76.8 | ||
+ | <tr><td>10x Pfu Buffer with MgSO<sub>4</sub><td>2<td>12 | ||
+ | <tr><td>dNTPs<td>1<td>6 | ||
+ | <tr><td>Standard Forward Primer<td>1<td>6 | ||
+ | <tr><td>Fusion Reverse Primer<td>1<td>6 | ||
+ | <tr><td>Template DNA<td>2<td>12 | ||
+ | <tr><td>Pfu polymerase<td>0.2<td>1.2 | ||
+ | </table><br> | ||
+ | |||
+ | Standard prefix has melting temperature of 68.7<sup>o</sup>C. Standard suffix has melting temperature of 61.6<sup>o</sup>C. | ||
+ | Annealing temperature of 56.6<sup>o</sup>C with 5 <sup>o</sup>C gradient. | ||
+ | |||
+ | PCR- Conditions: | ||
+ | 1. 95<sup>o</sup>C for 180 sec | ||
+ | 2. 95<sup>o</sup>C for 30 sec | ||
+ | 3. 56.6 +/- 5<sup>o</sup>C for 30 sec | ||
+ | 4. 72<sup>o</sup>C for 150 sec | ||
+ | 5. 72<sup>o</sup>C for 600 sec | ||
+ | (35 cycles) | ||
+ | |||
+ | ==<font color="white">September 18, 2010== | ||
+ | ===<font color="white">JS, DM=== | ||
+ | <b>Objective:</b> Overexpress mms6.<br> | ||
+ | Method: Followed overexpression protocol. One flask was induced with 250(µL) IPTG while another was induced with 50(µL). | ||
+ | |||
+ | <table><table border ="3"> | ||
+ | <tr><td><b>Time (minutes)</b></td><td><b>OD #1 (600λ)</b></td><td><b>OD #2 (600λ)</b></td></tr> | ||
+ | <tr><td>30</td><td>0.139</td><td>0.133</td></tr> | ||
+ | <tr><td>60</td><td>0.200</td><td>0.188</td></tr> | ||
+ | <tr><td>90</td><td>0.372</td><td>0.360</td></tr> | ||
+ | <tr><td>120</td><td>0.702</td><td>0.704</td></tr> | ||
+ | <tr><td>150(T<sub>0</sub>)</td><td>0.871</td><td>0.810</td></tr> | ||
+ | <tr><td>210(T<sub>1</sub>)</td><td>2.600</td><td>2.500</td></tr> | ||
+ | <tr><td>270(T<sub>2</sub>)</td><td>3.317</td><td>3.300</td></tr> | ||
+ | <tr><td>330(T<sub>3</sub>)</td><td>4.408</td><td>3.946</td></tr> | ||
+ | </table><br> | ||
+ | |||
+ | * Plated cells in presence of IPTG, Fe or IPTG and Fe on AMP(+) plates. Incubated at 37 <sup>o</sup>C. | ||
+ | ** 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 <sup>o</sup>C 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. | ||
+ | ---- | ||
+ | ===<font color="white">TF=== | ||
+ | |||
+ | <b>Objective:</b> Obtain preparations of B0015 (dT) and J04450 (RFP). | ||
+ | |||
+ | <b>Method:</b> Used [[Team:Lethbridge/Notebook/Protocols|Plasmid DNA Purification by Alkaline Lysis (Large Scale AKA Maxiprep]] protocol. | ||
+ | |||
+ | <b>Results:</b> Chloroform, DNA and isopropanol were mixed and therefore no products could be recovered. | ||
+ | ---- | ||
+ | |||
+ | |||
==<font color="white">September 20, 2010== | ==<font color="white">September 20, 2010== | ||
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<tr><td>8</td><td>RFP2</td><td>10</td><td>2</td></tr> | <tr><td>8</td><td>RFP2</td><td>10</td><td>2</td></tr> | ||
<tr><td>9</td><td>RFP3</td><td>10</td><td>2</td></tr></table><br> | <tr><td>9</td><td>RFP3</td><td>10</td><td>2</td></tr></table><br> | ||
- | <b>Results:</b | + | <b>Results:</b> <br> |
+ | [[image:100920.jpg|100px]] | ||
*mms6 was not amplified | *mms6 was not amplified | ||
*RFP was amplified | *RFP was amplified | ||
Line 318: | Line 582: | ||
***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. | ***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. | ||
---------------------------------------------------------------------------------------------------------------------- | ---------------------------------------------------------------------------------------------------------------------- | ||
- | <b>Objective:</b> Confirm assembly (3 antibiotic) of K118021 and B0015 from Sept. | + | <b>Objective:</b> Confirm assembly (3 antibiotic) of K118021 and B0015 from Sept. 14, 2010 via PCR.<br> |
<b>Method:</b> Set up 50uL reaction mixture with 2uL of ligated DNA. Used VF2 and VR primers.<br> | <b>Method:</b> Set up 50uL reaction mixture with 2uL of ligated DNA. Used VF2 and VR primers.<br> | ||
Used PFU setting on Thermocycler<br> | Used PFU setting on Thermocycler<br> | ||
- | <b>Results:</b | + | <b>Results:</b> <br> |
+ | [[image:100915Assembly.jpg|100px]] | ||
<b>Conclusion:</b> Assembly did not work as intended. | <b>Conclusion:</b> Assembly did not work as intended. | ||
+ | ---- | ||
+ | |||
+ | ===<font color="white">HB=== | ||
+ | |||
+ | <b>Objective:</b> PCR mms6 to attach fusion standard to the N-term and standard suffix to C-term.<br> | ||
+ | |||
+ | <table border ="3"> | ||
+ | <tr><td><b>Component</b><td><b>1X(µL)</b><td><b>Master Mix(x6)(µL)</b> | ||
+ | <tr><td>Milli-Q H<sub>2</sub>O<td>12.8<td>76.8 | ||
+ | <tr><td>10x Pfu Buffer with MgSO<sub>4</sub><td>2<td>12 | ||
+ | <tr><td>dNTPs<td>1<td>6 | ||
+ | <tr><td>Fusion Forward Primer<td>1<td>6 | ||
+ | <tr><td>Standard Reverse Primer<td>1<td>6 | ||
+ | <tr><td>Template DNA<td>2<td>12 | ||
+ | <tr><td>Pfu polymerase<td>0.2<td>1.2 | ||
+ | </table><br> | ||
+ | |||
+ | Prefix fusion sense primer has melting temperature of 56.1<sup>o</sup>C. Standard suffix primer has melting temperature of 61.3<sup>o</sup>C. | ||
+ | |||
+ | PCR- Conditions: | ||
+ | 1. 95<sup>o</sup>C for 180 sec | ||
+ | 2. 95<sup>o</sup>C for 30 sec | ||
+ | 3. 51.1<sup>o</sup>C for 30 sec | ||
+ | 4. 72<sup>o</sup>C for 150 sec | ||
+ | 5. 72<sup>o</sup>C for 600 sec | ||
+ | (35 cycles) | ||
+ | |||
+ | Program HBPREFU: | ||
+ | 1. 49.1<sup>o</sup>C 2. 50.2<sup>o</sup>C 3. 51.4<sup>o</sup>C 4. 52.6<sup>o</sup>C 5. 53.5<sup>o</sup>C | ||
==<font color="white">September 21, 2010== | ==<font color="white">September 21, 2010== | ||
Line 342: | Line 636: | ||
<b>Follow-up:</b><br> | <b>Follow-up:</b><br> | ||
Screen white colonies by addition of catechol to solution containing white cells. | Screen white colonies by addition of catechol to solution containing white cells. | ||
+ | ---- | ||
+ | ===<font color="white">TF, MC=== | ||
+ | |||
+ | <b>Objective:</b> Obtain a preparation of B0015 (dT). | ||
+ | |||
+ | <b>Method:</b> Used [[Team:Lethbridge/Notebook/Protocols|Plasmid DNA Purification by Alkaline Lysis (Large Scale AKA Maxiprep]] protocol. | ||
+ | ---- | ||
+ | ===<font color="white">AV=== | ||
+ | <b>Objective:</b> PCR amplify the signal sequences synthesized by Mr. Gene (K331007, K331008, K331009, K331012).<br> | ||
+ | |||
+ | Composition of each PCR tube: | ||
+ | <table><table border ="3"> | ||
+ | <tr><td><b>Component</b></td><td><b>Volume(µL)</b></td></tr> | ||
+ | <tr><td>10x Pfu Buffer w/ MgSO<sub>4</sub></td><td>2</td></tr> | ||
+ | <tr><td>dNTP (10mM)</td><td>1</td></tr> | ||
+ | <tr><td>VF2</td><td>1</td></tr> | ||
+ | <tr><td>VR</td><td>1</td></tr> | ||
+ | <tr><td>MilliQ H<sub>2</sub>O</td><td>12.8</td></tr> | ||
+ | <tr><td>Template DNA</td><td>2</td></tr> | ||
+ | <tr><td>Pfu DNA Polymerase</td><td>0.2</td></tr> | ||
+ | </table><br> | ||
+ | Used iGEM thermocycler setting: PFU.<br> | ||
+ | |||
==<font color="white">September 21, 2010== | ==<font color="white">September 21, 2010== | ||
===<font color="white">ADS=== | ===<font color="white">ADS=== | ||
Line 366: | Line 683: | ||
<tr><td>16</td><td>Empty</td><td></td><td></td></tr> | <tr><td>16</td><td>Empty</td><td></td><td></td></tr> | ||
<tr><td>17</td><td>Empty</td><td></td><td></td></tr></table><br> | <tr><td>17</td><td>Empty</td><td></td><td></td></tr></table><br> | ||
- | <b>Results:</b | + | <b>Results:</b> <br> |
+ | [[image:100921.jpg|200px]] | ||
*Both KG PCR (Standard-Fusion and Fusion-Standard) amplified | *Both KG PCR (Standard-Fusion and Fusion-Standard) amplified | ||
*ADS PCR Amplified, but no insert present. | *ADS PCR Amplified, but no insert present. | ||
Line 422: | Line 740: | ||
*4) Verify sequence | *4) Verify sequence | ||
*5) Submit to registry for sequencing | *5) Submit to registry for sequencing | ||
+ | ---- | ||
+ | ===<font color="white">TF, MC=== | ||
+ | |||
+ | <b>Objective:</b> Obtain a preparation of J04450 (RFP).<br> | ||
+ | |||
+ | <b>ethod:</b> Used [[Team:Lethbridge/Notebook/Protocols|Plasmid DNA Purification by Alkaline Lysis (Large Scale AKA Maxiprep]] protocol. | ||
==<font color="white">September 24, 2010== | ==<font color="white">September 24, 2010== | ||
Line 455: | Line 779: | ||
Cells containing plasmids were put into glycerol stocks and put into HJ's -80<sup>o</sup>C.<br> | Cells containing plasmids were put into glycerol stocks and put into HJ's -80<sup>o</sup>C.<br> | ||
+ | |||
+ | ===<font color="white">ADS=== | ||
+ | <b>Objective:</b> Move plasmid DNA made by JV (above, Sept 25, 2010) into pSB1C3 for submission to the Standard Registry of Parts.<br> | ||
+ | <b>Method:</b> | ||
+ | *1) Digest at 37<sup>o</sup> 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<sup>o</sup>C 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.<br> | ||
+ | <b>Results:</b> Obtained <font color="red">RESULT!!!</font color> positive colonies<br> | ||
+ | ---------------------------------------------------------------------------------------------------------------------- | ||
+ | <b>Objective:</b> Assemble KG's C-terminal fusion xylE (<partinfo>K331021</partinfo>) (<font color="red">UNCONFIRMED</font>) and C-terminal Arginine + dT (<partinfo>S04261</partinfo>) to create <partinfo>K331011</partinfo>. <br> | ||
+ | <b>Method:</b> | ||
+ | *1) Digest at 37<sup>o</sup> 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<sup>o</sup>C 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.<br> | ||
+ | <b>Results:</b> Obtained <font color="red">RESULT!!!</font color> positive colonies<br> | ||
+ | |||
+ | ==<font color="white">September 26, 2010== | ||
+ | ===<font color="white">ADS=== | ||
+ | <b>Objective:</b> Purify plasmid DNA from 5mL cultures grown from transformations of KG's ligated PCRs containing (<font color="red">UNCONFIRMED</font>) <partinfo>K331020</partinfo> and <partinfo>K331021</partinfo>.<br> | ||
+ | <b>Method:</b> Used Qiagen spin column method with BioBasic EZ-10 spin columns.<br> | ||
+ | ------------------------------------------------------------------------------------------------------------------------ | ||
+ | <b>Objective:</b> Assemble the following:<br> | ||
+ | *<partinfo>K331020</partinfo> + <partinfo>B0015</partinfo> to obtain <font color="red"> To be named</font> biobrick | ||
+ | *<partinfo>K331021</partinfo> + <partinfo>S04261</partinfo> to obtain <font color="red"> To be named</font> biobrick | ||
+ | <b>Method:</b><br> | ||
+ | *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<sup>o</sup>C for 10 min<br> | ||
+ | Heat kill at 80<sup>o</sup>C for 20 min <br> | ||
+ | Ligate with T4 Ligase for 10 min at room temperature<br> | ||
+ | Transform into DH5&alpha subcloning efficiency cells<br> | ||
+ | <b>Results:</b> Obtained <font color="red">RESULTS!!!!</font color> positive colonies.<br> | ||
+ | Also analyzed minipreps on 1.5% TAE Agarose Gel<br> | ||
+ | [[image:100926Minipreps.jpg|200px]] | ||
+ | |||
+ | ==<font color="white">September 29, 2010== | ||
+ | ===<font color="white">ADS=== | ||
+ | Received synthesized parts from Mr. GENE (all in pMA vectors)<br> | ||
+ | *<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<br> | ||
+ | Transform 1µL into DH5α subcloning efficiency cells<br> | ||
+ | Obtained TNTC colonies<br> | ||
+ | Started overnight 5mL cultures for miniprep and insertion into pSB1C3 vector. | ||
+ | |||
+ | ---- | ||
+ | ===<font color="white">AV=== | ||
+ | |||
+ | <b>Objective:</b> PCR the N-term fusion tag onto RFP (E1010) using sloppy annealing of the YFP/CFP fusion primers. <br><br> | ||
+ | |||
+ | Composition of each PCR tube: | ||
+ | <table><table border ="3"> | ||
+ | <tr><td><b>Component</b></td><td>Volume(µL)</td></tr> | ||
+ | <tr><td>10x Pfu Buffer w/ MgSO<sub>4</sub></td><td>2</td></tr> | ||
+ | <tr><td>dNTP (10mM)</td><td>1</td></tr> | ||
+ | <tr><td>N-term Fus Prefix</td><td>1</td></tr> | ||
+ | <tr><td>Biobrick Suffix</td><td>1</td></tr> | ||
+ | <tr><td>MilliQ H<sub>2</sub>O</td><td>12.8</td></tr> | ||
+ | <tr><td>Template DNA (J04450)</td><td>2</td></tr> | ||
+ | <tr><td>Pfu DNA Polymerase</td><td>0.2</td></tr> | ||
+ | </table><br> | ||
+ | |||
+ | PCR conditions: | ||
+ | <table><table border ="3"> | ||
+ | <tr><td><b>Step</b></td><td><b>Temperature (<sup>o</sup>C)</b></td><td><b>Time (mins)</b></td><td><b>Number of cycles</b></td></tr> | ||
+ | <tr><td>Initial Denaturation</td><td>95</td><td>2</td><td>1</td></tr> | ||
+ | <tr><td>Denaturation</td><td>95</td><td>0.5</td><td>25</td></tr> | ||
+ | <tr><td>Annealing</td><td>47.6, 50.5, 53.8 (gradient)</td><td>0.5</td><td>25</td></tr> | ||
+ | <tr><td>Extension</td><td>72</td><td>2</td><td>25</td></tr> | ||
+ | <tr><td>Final Extension</td><td>72</td><td>10</td><td>1</td></tr> | ||
+ | </table><br> | ||
+ | |||
+ | Results: Gel has not been run. Have decided to order RFP specific primers for next year.<br><br> | ||
+ | <br> |