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Team:SDU-Denmark/labnotes2
From 2010.igem.org
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''Notes:'' To samples were run at two different temperatures: 56,1˚C and 64,5˚C respectively. <br>Polymerase used: Pfu<br> Primers used: None, as the two strands are supposed to anneal to each other<br><br> | ''Notes:'' To samples were run at two different temperatures: 56,1˚C and 64,5˚C respectively. <br>Polymerase used: Pfu<br> Primers used: None, as the two strands are supposed to anneal to each other<br><br> | ||
''Results:''<br><br> | ''Results:''<br><br> | ||
| + | |||
=== Gel extraction of flhD/C === | === Gel extraction of flhD/C === | ||
''Start date:'' 19/07 ''End date:'' 19/07<br> | ''Start date:'' 19/07 ''End date:'' 19/07<br> | ||
| Line 65: | Line 66: | ||
Nanodrop measurements idicated a possible contamination. However the DNA was pooled and used for digestion <br> | Nanodrop measurements idicated a possible contamination. However the DNA was pooled and used for digestion <br> | ||
--[[User:Tipi|Tipi]] 17:22, 19 July 2010 (UTC) | --[[User:Tipi|Tipi]] 17:22, 19 July 2010 (UTC) | ||
| + | === Digestion of flhD/C and pSB1A2 with EcoRI SpeI === | ||
| + | ''Start date:'' 19/07 ''End date:'' 19/07<br> | ||
| + | ''Methods:'' Digestion, Gel electrophoresis <br> | ||
| + | ''Protocol:''[https://2010.igem.org/Team:SDU-Denmark/protocols#Restriction_digest RD1.1] | ||
| + | ''Experiment done by:''Maria <br><br> | ||
| + | ''Notes:'' <br> | ||
| + | purified pSB1C3 (tube 18 blue) and flhD/C from [https://2010.igem.org/Team:SDU-Denmark/labnotes2#Gel_extraction_of_flhD.2FC Gel extraction] was digested with EcoRI and SpeI.<br> | ||
| + | Restriction mixture: <br> | ||
| + | |||
| + | <table style="text-align: left; width: 100px;" border="1" | ||
| + | cellpadding="2" cellspacing="2"> | ||
| + | <tr> | ||
| + | <td style="vertical-align: top;"><br> | ||
| + | </td> | ||
| + | <td style="vertical-align: top;">flhD/C<br> | ||
| + | </td> | ||
| + | <td style="vertical-align: top;">pSB1C3<br> | ||
| + | </td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td style="vertical-align: top;">H2O<br> | ||
| + | </td> | ||
| + | <td style="vertical-align: top;">30uL<br> | ||
| + | </td> | ||
| + | <td style="vertical-align: top;">24uL<br> | ||
| + | </td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td style="vertical-align: top;">EcoRI<br> | ||
| + | </td> | ||
| + | <td style="vertical-align: top;">4uL<br> | ||
| + | </td> | ||
| + | <td style="vertical-align: top;">2uL<br> | ||
| + | </td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td style="vertical-align: top;">SpeI<br> | ||
| + | </td> | ||
| + | <td style="vertical-align: top;">4uL<br> | ||
| + | </td> | ||
| + | <td style="vertical-align: top;">2uL<br> | ||
| + | </td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td style="vertical-align: top;">FD green buffer<br> | ||
| + | </td> | ||
| + | <td style="vertical-align: top;">8uL<br> | ||
| + | </td> | ||
| + | <td style="vertical-align: top;">4uL<br> | ||
| + | </td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td style="vertical-align: top;">DNA<br> | ||
| + | </td> | ||
| + | <td style="vertical-align: top;">38uL<br> | ||
| + | </td> | ||
| + | <td style="vertical-align: top;">10uL<br> | ||
| + | </td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td style="vertical-align: top;">total vol.<br> | ||
| + | </td> | ||
| + | <td style="vertical-align: top;">84<br> | ||
| + | </td> | ||
| + | <td style="vertical-align: top;">42<br> | ||
| + | </td> | ||
| + | </tr> | ||
| + | </table> | ||
| + | <br><br> | ||
| + | ''Results:''<br> | ||
| + | Gel electrophoresis:<br> | ||
| + | [[Image:Team-SDU-Denmark-FlhD,C digestion with SpeI and EcoRI.jpg |600px]]<br><br> | ||
| + | ''Analysis:''<br> | ||
| + | In lane 2 containing pSB1C3 2 bands are detected indicating a succesful digestion of the plasmid (the band at 1000 bp corresponds to J04450). A succesful digestion of the flhD/C cannot be concluded from the gel. However both bands was excised and extracted from gel <br> | ||
| + | --[[User:Tipi|Tipi]] 17:42, 19 July 2010 (UTC) | ||
== Group: Photosensor == | == Group: Photosensor == | ||
Revision as of 17:42, 19 July 2010
Lab notes (7/19 - 7/25)
Contents |
Group: Flagella
Annealing of the two mutated strands of FlhDC
Experiment done by: Sheila
Date: July 19th
Protocol: CP1.1
Method: PCR of the two mutated strands of the FlhDC operon
Notes: To samples were run at two different temperatures: 56,1˚C and 64,5˚C respectively.
Polymerase used: Pfu
Primers used: None, as the two strands are supposed to anneal to each other
Results:
Gel extraction of flhD/C
Start date: 19/07 End date: 19/07
Methods: Gel extraction, nanodrop
Protocol:DE1.1
Experiment done by:Maria
Notes:
70uL of flhD/C PCR product from Amplification of flhD/C was loaded onto a 1.5 agarose gel and extracted according to protocol.
DNA was eluted in 20uL elution buffer.
Results:
Nanodrop:
| Sample ID |
ng/uL |
260/280 |
260/230 |
| flhD/C 1 |
19.82 |
2.43 |
0.08 |
| flhD/C 2 |
25.24 |
2.22 |
0.12 |
Analysis:
Nanodrop measurements idicated a possible contamination. However the DNA was pooled and used for digestion
--Tipi 17:22, 19 July 2010 (UTC)
Digestion of flhD/C and pSB1A2 with EcoRI SpeI
Start date: 19/07 End date: 19/07
Methods: Digestion, Gel electrophoresis
Protocol:RD1.1
Experiment done by:Maria
Notes:
purified pSB1C3 (tube 18 blue) and flhD/C from Gel extraction was digested with EcoRI and SpeI.
Restriction mixture:
| flhD/C |
pSB1C3 |
|
| H2O |
30uL |
24uL |
| EcoRI |
4uL |
2uL |
| SpeI |
4uL |
2uL |
| FD green buffer |
8uL |
4uL |
| DNA |
38uL |
10uL |
| total vol. |
84 |
42 |
Results:
Gel electrophoresis:
Analysis:
In lane 2 containing pSB1C3 2 bands are detected indicating a succesful digestion of the plasmid (the band at 1000 bp corresponds to J04450). A succesful digestion of the flhD/C cannot be concluded from the gel. However both bands was excised and extracted from gel
--Tipi 17:42, 19 July 2010 (UTC)
Group: Photosensor
Group: Retinal
Transformation of K081005 in pSB1A2 (constitutive promoter and RBS combined),R0011 in pSB1A2, pSB3C5 w. J04450 and pSB3T5 w. J04450 in Top 10 E.Coli
Start date: 19/07 End date: 20/07
Methods: ON culture, making competent cells, transformation
Protocol:CC1.1 TR1.1
Experiment done by: Maria
Notes:ON colony was made of 110 ml lb medium inoculated with a top10 coloni.
| Time |
Optical density |
| 8:12 |
2.9 |
| 8:17 |
0.02 |
| 9:17 |
0.035 |
| 10:17 |
0.204 |
| 10:40 |
0.380 |
| 10:50 |
0.49 |
pSB1A2 w. R0011 and pSB1A2 w. K081005 was plated with 150uL on plates containing LA, LA + Amp, LA + Tetracycline, LA + Chloramphenicol and LA + Kanamycine.Upconcentration of these samples was not made. pSB3T5 w. J04450 and pSB3C5 w. J04450 was plated according to protocol
Results:
Analysis:
--Tipi 16:33, 19 July 2010 (UTC)
Include column content here.
