Team:SDU-Denmark/labnotes

From 2010.igem.org

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''Protocol'': [https://2010.igem.org/Team:SDU-Denmark/protocols#CP1.2 CP1.2] <br><br>
''Protocol'': [https://2010.igem.org/Team:SDU-Denmark/protocols#CP1.2 CP1.2] <br><br>
''Notes'': The plasmids were collected from tubes green 10 and green 11 in the freezer. VR and VF2 primers were used.<br>
''Notes'': The plasmids were collected from tubes green 10 and green 11 in the freezer. VR and VF2 primers were used.<br>
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The experiment were performed simultaneously with the Taq control version of [https://2010.igem.org/Team:SDU-Denmark/labnotes#Extraction_of_BBa_B0034_from_pSB1A2_plasmid_using_PFU the extraction of BBa_B0034 from pSB1A2 plasmid] done by the phototaxis goup  <br><br>
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The experiment were performed simultaneously with the Taq control version of [https://2010.igem.org/Team:SDU-Denmark/labnotes#Extraction_of_B0034_from_pSB1A2_plasmid_using_PFU the extraction of B0034 from pSB1A2 plasmid] done by the phototaxis goup  <br><br>
--[[User:Sheila|Sheila]] 09:08, 14 July 2010 (UTC)
--[[User:Sheila|Sheila]] 09:08, 14 July 2010 (UTC)
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== Group: Retinal ==
== Group: Retinal ==
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=== Transformation of TOP10 ''e. coli'' with [http://partsregistry.org/Part:BBa_K274210 BBa_K274210]===
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=== Transformation of TOP10 ''e. coli'' with [http://partsregistry.org/Part:BBa_K274210 K274210]===
<br>
<br>
Experiment continued from last week.
Experiment continued from last week.
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Gel was loaded with DNA-ladder plus, with the upper marker at 5000 bp.<br><br>
Gel was loaded with DNA-ladder plus, with the upper marker at 5000 bp.<br><br>
''Results:'' We found plasmids at the expected 5 kb marker in colonies 1-8. Successfull plates were kept in incubator over night. They will be made into overnight cultures and thereafter frozen.<br><br>
''Results:'' We found plasmids at the expected 5 kb marker in colonies 1-8. Successfull plates were kept in incubator over night. They will be made into overnight cultures and thereafter frozen.<br><br>
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''Analysis:'' It seems we have transformed our cells with BBa_K274210. Confirmation will come when we run experiments to demonstrate presence of beta-carotene. <br><br>
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''Analysis:'' It seems we have transformed our cells with K274210. Confirmation will come when we run experiments to demonstrate presence of beta-carotene. <br><br>
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=== Transformation of [http://partsregistry.org/Part:BBa_R0011 BBa_R0011 ]and [http://partsregistry.org/Part:BBa_I0500 BBa_I0500] ===
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=== Transformation of [http://partsregistry.org/Part:BBa_R0011 R0011 ]and [http://partsregistry.org/Part:BBa_I0500 I0500] ===
Start date: July 12th    End date:July 14th<br>
Start date: July 12th    End date:July 14th<br>
''Methods:'' Overnight culture, competent cells, transformation.<br>
''Methods:'' Overnight culture, competent cells, transformation.<br>
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Analysis: We suspect the agar dishes might have been the cause of this.<br><br>
Analysis: We suspect the agar dishes might have been the cause of this.<br><br>
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=== Mini-prep of pSB1A2 w. [http://partsregistry.org/Part:BBa_B0034 BBa_B0034], pSB1AK3 w. [http://partsregistry.org/Part:BBa_B0015 BBa_B0015] and pSB3K3 w. [http://partsregistry.org/Part:BBa_J04450 BBa_J04450] (transformation from 08/07) ===
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=== Mini-prep of pSB1A2 w. [http://partsregistry.org/Part:BBa_B0034 B0034], pSB1AK3 w. [http://partsregistry.org/Part:BBa_B0015 B0015] and pSB3K3 w. [http://partsregistry.org/Part:BBa_J04450 J04450] (transformation from 08/07) ===
Start date: July 12th<br>
Start date: July 12th<br>
''Methods:'' Mini-prep  <br><br>
''Methods:'' Mini-prep  <br><br>
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All bands appeared to be about 1000 kb smaller than the expected size. This could be due to the supercoiling of the plasmids. To verify this, the plasmids were cut with PstI: [https://2010.igem.org/Team:SDU-Denmark/labnotes#Digestion_of_pSB1A2_w._B0034.2C_pSB1AK3_w._B0015_and_pSB3K3_w._J04450_and_pSB1A2_using_pstI.28miniprep_from_12.2F07.29 Digestion of plasmids]
All bands appeared to be about 1000 kb smaller than the expected size. This could be due to the supercoiling of the plasmids. To verify this, the plasmids were cut with PstI: [https://2010.igem.org/Team:SDU-Denmark/labnotes#Digestion_of_pSB1A2_w._B0034.2C_pSB1AK3_w._B0015_and_pSB3K3_w._J04450_and_pSB1A2_using_pstI.28miniprep_from_12.2F07.29 Digestion of plasmids]
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=== Digestion of pSB1A2 w. [http://partsregistry.org/Part:BBa_B0034 BBa_B0034], pSB1AK3 w. [http://partsregistry.org/Part:BBa_B0015 BBa_B0015] and pSB3K3 w. [http://partsregistry.org/Part:BBa_J04450 BBa_J04450]and pSB1A2 using pstI(miniprep from 12/07) ===
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=== Digestion of pSB1A2 w. [http://partsregistry.org/Part:BBa_B0034 B0034], pSB1AK3 w. [http://partsregistry.org/Part:BBa_B0015 B0015] and pSB3K3 w. [http://partsregistry.org/Part:BBa_J04450 J04450]and pSB1A2 using pstI(miniprep from 12/07) ===
Start date: July 13th<br>
Start date: July 13th<br>
''Methods:'' Digestion  <br>
''Methods:'' Digestion  <br>
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--[[User:Tipi|Tipi]] 15:53, 19 July 2010 (UTC)
--[[User:Tipi|Tipi]] 15:53, 19 July 2010 (UTC)
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=== Transformation of ''E. coli'' MG1655 with [http://partsregistry.org/Part:BBa_K274210 BBa_K274210], [http://partsregistry.org/Part:BBa_E0040 BBa_E0040] and [http://partsregistry.org/Part:BBa_I0500 BBa_I0500] ===
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=== Transformation of ''E. coli'' MG1655 with [http://partsregistry.org/Part:BBa_K274210 K274210], [http://partsregistry.org/Part:BBa_E0040 E0040] and [http://partsregistry.org/Part:BBa_I0500 I0500] ===
Start Date: July 13th    <br>
Start Date: July 13th    <br>
''Methods:'' overnight cultures, Competent Cells, Transformation of competent cells. <br>
''Methods:'' overnight cultures, Competent Cells, Transformation of competent cells. <br>
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''Notes:'' Growth was started at 09.30 with an OD550 of 0.19A. The cells were harvested at 10.26 where OD550 was 0.48.<br><br>
''Notes:'' Growth was started at 09.30 with an OD550 of 0.19A. The cells were harvested at 10.26 where OD550 was 0.48.<br><br>
''Results:'' Competent cells were made according to protocol. Apparently ''E. coli'' MG1655 reaches OD550 0.5 at least an hour faster than ''E. coli'' TOP10.<br><br>
''Results:'' Competent cells were made according to protocol. Apparently ''E. coli'' MG1655 reaches OD550 0.5 at least an hour faster than ''E. coli'' TOP10.<br><br>
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'''Experiment:''' Transforming ''E.coli'' MG1655 strain with BBa_E0040 (GFP coding sequence) and BBa_I0500 (pBad arabinose inducible promoter)
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'''Experiment:''' Transforming ''E.coli'' MG1655 strain with E0040 (GFP coding sequence) and I0500 (pBad arabinose inducible promoter)
<br>
<br>
Date: July 13th<br><br>
Date: July 13th<br><br>
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Only 1 microliter DNA material was transferred per tube, since it was taken from the iGEM 2010 distribution plates.<br><br>
Only 1 microliter DNA material was transferred per tube, since it was taken from the iGEM 2010 distribution plates.<br><br>
Plates were dried at 42°C for 15 minutes, just prior to plating. Upon plating we discovered a problem with many of the ampicilin plates we made a couple of days ago. The agar breaks on the slightest contact, and are therfore impossible to use.<br><br>
Plates were dried at 42°C for 15 minutes, just prior to plating. Upon plating we discovered a problem with many of the ampicilin plates we made a couple of days ago. The agar breaks on the slightest contact, and are therfore impossible to use.<br><br>
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As a result of the defective agar plates, most of our cells carrying BBa_E0040 were ruined. We managed to save and plate out two batches, one from each tube, so we are hoping for the best tomorrow.<br><br>
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As a result of the defective agar plates, most of our cells carrying E0040 were ruined. We managed to save and plate out two batches, one from each tube, so we are hoping for the best tomorrow.<br><br>
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Results: Only cells with BBa_E0040 grew colonies by overnight incubation.
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Results: Only cells with E0040 grew colonies by overnight incubation.
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=== Miniprep of [http://partsregistry.org/Part:BBa_K098995 BBa_K098995] in pSB1A2 ===
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=== Miniprep of [http://partsregistry.org/Part:BBa_K098995 K098995] in pSB1A2 ===
Start date: July 13th    <br>
Start date: July 13th    <br>
''Methods:''  Fermentas GeneJET plasmid miniprep kit <br><br>
''Methods:''  Fermentas GeneJET plasmid miniprep kit <br><br>
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''Analysis: ''All four samples turned out fine, so they were pooled and frozen. (See results for details)
''Analysis: ''All four samples turned out fine, so they were pooled and frozen. (See results for details)
<br>
<br>
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Nr. 28 in the freezer = BBa_K098995<br><br>
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Nr. 28 in the freezer = K098995<br><br>
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=== Extraction of [http://partsregistry.org/Part:BBa_B0034 BBa_B0034] from pSB1A2 plasmid using PFU ===
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=== Extraction of [http://partsregistry.org/Part:BBa_B0034 B0034] from pSB1A2 plasmid using PFU ===
==== Preliminary PCR to amplify VF2-VR piece containing part and restriction sites ====
==== Preliminary PCR to amplify VF2-VR piece containing part and restriction sites ====
Date: July 14th <br>
Date: July 14th <br>
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--[[User:CKurtzhals|CKurtzhals]] 16:07, 15 July 2010 (UTC)
--[[User:CKurtzhals|CKurtzhals]] 16:07, 15 July 2010 (UTC)
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=== Miniprep and transformation of [http://partsregistry.org/Part:BBa_K274210 BBa_K274210] in pSB1A2 ===
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=== Miniprep and transformation of [http://partsregistry.org/Part:BBa_K274210 K274210] in pSB1A2 ===
Start date: July 14.th <br>
Start date: July 14.th <br>
''Methods:''  Fermentas GeneJET plasmid miniprep kit <br><br>
''Methods:''  Fermentas GeneJET plasmid miniprep kit <br><br>
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'''Transformation of the miniprepped plasmid:'''<br><br>
'''Transformation of the miniprepped plasmid:'''<br><br>
''Experiment done by:'' LC, Maria <br><br>  ''Protocol:'' TR 1.1 <br><br>
''Experiment done by:'' LC, Maria <br><br>  ''Protocol:'' TR 1.1 <br><br>
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''Notes:'' Transformed BBa_K274210 in pSB1A2 on LA plates with 100 microgram/ml ampicillin into ''E. coli'' MG1655. We used 2 microliter from the miniprep product for each sample.<br>
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''Notes:'' Transformed K274210 in pSB1A2 on LA plates with 100 microgram/ml ampicillin into ''E. coli'' MG1655. We used 2 microliter from the miniprep product for each sample.<br>
Our suspicion that the agar plates we made are faulty got confirmed, three more plates were destroyed while spreading the bacteria on them (Sample 1a, 2b, 2pellet and the positive control)<br><br>
Our suspicion that the agar plates we made are faulty got confirmed, three more plates were destroyed while spreading the bacteria on them (Sample 1a, 2b, 2pellet and the positive control)<br><br>
''Results:'' Cells grew just fine and the negative control was as expected. After a night at 37° the cells seemed a little more yellowish to the eye, than the usual. <br><br>
''Results:'' Cells grew just fine and the negative control was as expected. After a night at 37° the cells seemed a little more yellowish to the eye, than the usual. <br><br>
''Analysis:'' The transformations succeeded. <br><br>
''Analysis:'' The transformations succeeded. <br><br>
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=== Coloni PCR of pSB1A2 w. [http://partsregistry.org/Part:BBa_E0040 BBa_E0040] (transformation 13/7) ===
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=== Coloni PCR of pSB1A2 w. [http://partsregistry.org/Part:BBa_E0040 E0040] (transformation 13/7) ===
Start date: July 14th <br>
Start date: July 14th <br>
''Methods:''  Taq coloni PCR and gel electrophoresis <br><br>
''Methods:''  Taq coloni PCR and gel electrophoresis <br><br>
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--[[User:Tipi|Tipi]] 16:09, 19 July 2010 (UTC)
--[[User:Tipi|Tipi]] 16:09, 19 July 2010 (UTC)
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=== Transformation of [http://partsregistry.org/Part:BBa_K081005 BBa_K081005] in pSB1A2 (constitutive promoter and RBS combined) and [http://partsregistry.org/Part:BBa_R0011 BBa_R0011] in pSB1A2 in Top 10 E.Coli ===
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=== Transformation of [http://partsregistry.org/Part:BBa_K081005 K081005] in pSB1A2 (constitutive promoter and RBS combined) and [http://partsregistry.org/Part:BBa_R0011 R0011] in pSB1A2 in Top 10 E.Coli ===
Start date: July 15th - 16th <br>
Start date: July 15th - 16th <br>
''Methods:''  Overnight culture, making competent cells, transformation <br><br>
''Methods:''  Overnight culture, making competent cells, transformation <br><br>
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--[[User:Lclund|Lclund]] 10:00, 19 July 2010 (UTC)
--[[User:Lclund|Lclund]] 10:00, 19 July 2010 (UTC)
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==== Restriction digest of [http://partsregistry.org/Part:BBa_E0040 BBa_E0040] miniprep product ====
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==== Restriction digest of [http://partsregistry.org/Part:BBa_E0040 E0040] miniprep product ====
Start date: July 16th<br>
Start date: July 16th<br>
''Methods:''  Restriction digest <br><br>
''Methods:''  Restriction digest <br><br>
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<br><br>
<br><br>
''Analysis:'' Sample 2 and 3 got pooled and frozen, 1 and 4 were discarded, since their length didn't seem exactly right.<br><br>
''Analysis:'' Sample 2 and 3 got pooled and frozen, 1 and 4 were discarded, since their length didn't seem exactly right.<br><br>
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Nr. 12 in the freezer = BBa_E0040 miniprep product in pSB1A2.<br>
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Nr. 12 in the freezer = E0040 miniprep product in pSB1A2.<br>
--[[User:Lclund|Lclund]] 10:00, 19 July 2010 (UTC)
--[[User:Lclund|Lclund]] 10:00, 19 July 2010 (UTC)

Latest revision as of 19:16, 24 October 2010