Team:Caltech/Week 5
From 2011.igem.org
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__NOTOC__ | __NOTOC__ | ||
==July 11== | ==July 11== | ||
+ | [[File:7-11gelpNT003assem.jpg|thumb|left|1 ladder, 2 R0010, 3 K123003, 4 B0014, 5 pSB4A5]] | ||
<p>Miniprep pSB4A5<br/> | <p>Miniprep pSB4A5<br/> | ||
Run PCR to amplify [http://partsregistry.org/Part:BBa_R0010 R0010], [http://partsregistry.org/Part:BBa_K123003 K123003], [http://partsregistry.org/Part:BBa_B0014 B0014] and [http://partsregistry.org/Part:BBa_PSB4A5 PSB4A5]<br/> | Run PCR to amplify [http://partsregistry.org/Part:BBa_R0010 R0010], [http://partsregistry.org/Part:BBa_K123003 K123003], [http://partsregistry.org/Part:BBa_B0014 B0014] and [http://partsregistry.org/Part:BBa_PSB4A5 PSB4A5]<br/> | ||
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===Results=== | ===Results=== | ||
The PCR of parts for pNT003 was successful except for the terminator (B0014), as there is no band in that lane. | The PCR of parts for pNT003 was successful except for the terminator (B0014), as there is no band in that lane. | ||
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==July 12== | ==July 12== | ||
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[[File:7-12pcrgel.jpg|thumb|from left to right: 1 ladder, 2 R0010, 3 K123000, 4 B0014 for pNT002, 5 R0040, 6 K123001, 7 B0014 for pNT003, 8 B0014 for pNT001]] | [[File:7-12pcrgel.jpg|thumb|from left to right: 1 ladder, 2 R0010, 3 K123000, 4 B0014 for pNT002, 5 R0040, 6 K123001, 7 B0014 for pNT003, 8 B0014 for pNT001]] | ||
+ | Transformed competent cells with pSB3C5.<br/> | ||
+ | Ran PCR of parts for pNT001 and pNT002 Gibson assembly.<br/> | ||
+ | Started pulsed field gel electrophoresis of some of our LA River DNA obtained by the Mo Bio kits in week 2.<br/> | ||
+ | ===Results=== | ||
+ | All of the parts for pNT001, pNT002 and B0014 were PCR'd with primers for Gibson assembly. All lanes except for lanes 4 and 5 had single bands, indicating that the parts were successfully amplified. | ||
+ | |||
+ | ==July 13== | ||
+ | Retrieved and imaged pulsed field gel.<br/> | ||
+ | Ran PCR of R0040 and B0014 with primers that failed yesterday.<br/> | ||
+ | Purified PCR products for Gibson of pNT001 and pNT003.<br/> | ||
+ | Started overnights of pSB3C5.<br/> | ||
+ | Replated some pSB3C5<br/> | ||
+ | ===Results=== | ||
+ | <p>The pulsed field gel had to be restained after running. The ladder and control appeared, but none of our LA River sample DNA. Since the ladder ranges from 5kb to 50 kb, we should be able to see some smears in the experimental lanes, as we ran a short gel last week and found that the sample DNA was greater than 10kb. Since the ladder does not seem well separated, we will try running the gel again with different parameters.</p> | ||
+ | The pSB3C5 had many colonies, but none of them are red. This is strange because the plasmid contains BBa_J04450 as an insert, a RFP. We are replating on a new chlor plate and starting overnights to send off for sequencing to make sure the plasmid is okay.<br/> | ||
+ | We redid the PCR of R0040 and B0014, but no bands appeared in the gel.<br/> | ||
+ | [[File:7-13pulsedgel.jpg|thumb|lanes 1 lambda mono cut ladder; 2 blank; 1 fosmid kit control (100 ng); 9-1 Part(2000 ng); 10-1 Part(300 ng); 4 LA River location 9; LA River location 10]] | ||
+ | PCR Purifications for Gibson | ||
+ | <table border="1"> | ||
+ | <tr> | ||
+ | <th>Part</th> | ||
+ | <th>Concentration (ng/ul)</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>R0010 for pNT003</td> | ||
+ | <td>130.5</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>K123003</td> | ||
+ | <td>99.9</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>pSB4A5 + prefix and suffix</td> | ||
+ | <td>90.4</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>R0010 for pNT001</td> | ||
+ | <td>142.5</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>K123000</td> | ||
+ | <td>111.4</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>K123001</td> | ||
+ | <td>123.0</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>B0014 for pNT003</td> | ||
+ | <td>119.9</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>B0014 for pNT001</td> | ||
+ | <td>139.7</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | |||
+ | ==July 14== | ||
+ | <p>Miniprep overnights and send off for sequencing<br/> | ||
+ | Begin 16S sequencing by PCRing the samples with 16s universal primers and the vector we are using<br/> | ||
+ | Try to PCR the miniprep DNA with the pSB3C5 primers<br/> | ||
+ | Do Gibson with the PCR parts that worked</p> | ||
+ | |||
+ | '''Labels for 16s sequencing PCR:''' | ||
+ | * 9-1 (1/10): 9a | ||
+ | * 9-1 (1/100): 9b | ||
+ | * 9-1 (1/1000): 9c | ||
+ | * 10-3 (1/10): 10a | ||
+ | * 10-3 (1/100): 10b | ||
+ | * 10-3 (1/1000): 10c | ||
+ | * Control 1: C1 | ||
+ | * Control 2: C2 | ||
+ | |||
+ | '''Labels for PCR Miniprep:''' | ||
+ | * pSB3C5 (1/100)- HA | ||
+ | * pSB3C5 (1/100)- HB | ||
+ | * pSB3C5 (1/100)- HC | ||
+ | * pSB3C5 (1/1000)- TA | ||
+ | * pSB3C5 (1/1000)- TB | ||
+ | * pSB3C5 (1/1000)- TC | ||
+ | |||
+ | ===Results=== | ||
+ | The minipreps of the overnight cultures all had very low concentrations of DNA. | ||
+ | <table border="1"> | ||
+ | <tr> | ||
+ | <th>Miniprep</th> | ||
+ | <th>Concentration (ng/ul)</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>PSB3C5 A</td> | ||
+ | <td>3.1</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>PSB3C5 B</td> | ||
+ | <td>4.0</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>PSB3C5 C</td> | ||
+ | <td>3.6</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | <gallery> | ||
+ | File:Gel 2 7-14.JPG|lane 1 ladder; 2 sample 9; 3 sample 9; 4 sample 9; 5 sample 10; 6 sample 10; 7 sample 10; 8 negative control | ||
+ | File:Gel 3 7-14.JPG|lane 1 ladder; 2-7 pSB3C5 minipreps, differing dilutions | ||
+ | File:Gel 4 7-14.JPG|ane 1 ladder; 2-4 pET53EST (V1, V2, V3) | ||
+ | </gallery> | ||
+ | ==July 15== | ||
+ | Run gel of LA River samples. Perhaps the freezing and thawing has degraded the DNA further.<br/> | ||
+ | Redo PCR of B0014 and R0040 with different annealing temperatures.<br/> | ||
+ | Redo Gibson of pNT001 and pNT003, doing 10 ul reactions, transform into Joe's competent cells<br/> | ||
+ | Make kanamycin stock and plates.<br/> | ||
+ | Gibson assemble 16s with the vector<br/> | ||
+ | Transform cells with pSB3K5, because we can't use pSB3C5 in XL-10 Golds<br/> | ||
+ | ===Results=== | ||
+ | Gibson Plates | ||
+ | <table border="1"> | ||
+ | <tr> | ||
+ | <th>Plate</th> | ||
+ | <th>Number of Colonies</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>pNT001 +</td> | ||
+ | <td>0</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>pNT001 -</td> | ||
+ | <td>0</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>pNT003 +</td> | ||
+ | <td>0</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>pNT003 -</td> | ||
+ | <td>0</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>vector (pSB4A5 only)</td> | ||
+ | <td>0</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | Perhaps our cells are not being very competent again. We should do a 20 ul reaction instead of a 10.5 ul reaction so we can retransform if necessary. Also, we should change amounts of DNA in the reaction. We've been diluting the DNA and trying to get all the parts within a 10 ng range. <br/> | ||
+ | [[File:7-15larivergel.jpg|thumb|Lane 1 ladder, 2-4 sample 9, 5-7 sample 10]] | ||
+ | The LA River DNA has been degraded to be a smear between .1-10kb, too short to use in the pulsed field gel and the fosmid kit. | ||
+ | ==July 16== | ||
+ | ===Results=== | ||
+ | <p>We are getting a lot of self-ligation of the vector.<br/> | ||
+ | Joe's cells are more competent than ours. We used our XL-10 Golds to transform pSB3K5 and had 0 colonies in the plate. We will need to redo the transformation.</p><br/> | ||
+ | Gibson Plates | ||
+ | <table border="1"> | ||
+ | <tr> | ||
+ | <th>Plate</th> | ||
+ | <th>Number of Colonies</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>pNT001 +</td> | ||
+ | <td>6</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>pNT001 -</td> | ||
+ | <td>8</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>pNT003 +</td> | ||
+ | <td>0</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>pNT003 -</td> | ||
+ | <td>12</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>vector (pSB4A5 only)</td> | ||
+ | <td>76</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>16s sample 9a</td> | ||
+ | <td>0</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>16s sample 9b</td> | ||
+ | <td>0</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>16s sample 10a</td> | ||
+ | <td>1</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>16s sample 10b</td> | ||
+ | <td>2</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>16s vector only (pET53DEST)</td> | ||
+ | <td>1</td> | ||
+ | </tr></table> | ||
}} | }} |
Latest revision as of 18:25, 20 July 2011
Project |
July 11Miniprep pSB4A5 ResultsThe PCR of parts for pNT003 was successful except for the terminator (B0014), as there is no band in that lane. July 12Transformed competent cells with pSB3C5. ResultsAll of the parts for pNT001, pNT002 and B0014 were PCR'd with primers for Gibson assembly. All lanes except for lanes 4 and 5 had single bands, indicating that the parts were successfully amplified. July 13Retrieved and imaged pulsed field gel. ResultsThe pulsed field gel had to be restained after running. The ladder and control appeared, but none of our LA River sample DNA. Since the ladder ranges from 5kb to 50 kb, we should be able to see some smears in the experimental lanes, as we ran a short gel last week and found that the sample DNA was greater than 10kb. Since the ladder does not seem well separated, we will try running the gel again with different parameters. The pSB3C5 had many colonies, but none of them are red. This is strange because the plasmid contains BBa_J04450 as an insert, a RFP. We are replating on a new chlor plate and starting overnights to send off for sequencing to make sure the plasmid is okay. PCR Purifications for Gibson
July 14Miniprep overnights and send off for sequencing Labels for 16s sequencing PCR:
Labels for PCR Miniprep:
ResultsThe minipreps of the overnight cultures all had very low concentrations of DNA.
July 15Run gel of LA River samples. Perhaps the freezing and thawing has degraded the DNA further. ResultsGibson Plates
Perhaps our cells are not being very competent again. We should do a 20 ul reaction instead of a 10.5 ul reaction so we can retransform if necessary. Also, we should change amounts of DNA in the reaction. We've been diluting the DNA and trying to get all the parts within a 10 ng range. The LA River DNA has been degraded to be a smear between .1-10kb, too short to use in the pulsed field gel and the fosmid kit. July 16ResultsWe are getting a lot of self-ligation of the vector. Gibson Plates
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