Team:SDU-Denmark/labnotes3

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(Restriction digest on miniprepped pOT2 plasmids with ninaB insert using EcoRI and XhoI)
(colony PCR using taq on ligated BBa_J13002 in pSB1C3)
 
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Results: A band showed at around 300bp in well 4.1. The expected length was 312bp. wells3.3 and 3.4 show a band outside the blue ladder, that could be rfp (since this was the original insert.) tube 3.2 was ruined.<br>
Results: A band showed at around 300bp in well 4.1. The expected length was 312bp. wells3.3 and 3.4 show a band outside the blue ladder, that could be rfp (since this was the original insert.) tube 3.2 was ruined.<br>
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[[Image:Team-sdu-denmark-Promotor_Rbs_i_psb3t5_colonipcr.jpg | 400px]]
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[[Image:Team-sdu-denmark-Promotor_Rbs_i_psb3t5_colonipcr.jpg | 300px]]
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<br><br>
Analysis: Well 3.2 shows promising results. ON and miniprep will be made, and it will be sent for sequencing.<br><br>
Analysis: Well 3.2 shows promising results. ON and miniprep will be made, and it will be sent for sequencing.<br><br>
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''Notes:'' purified flhD/C and miniprep of pSB1C3 and pSB3K3 were digested using the EcoRI and PstI restriction enzymes. <br><br>
''Notes:'' purified flhD/C and miniprep of pSB1C3 and pSB3K3 were digested using the EcoRI and PstI restriction enzymes. <br><br>
Restriction mixture (FlhD/C):<br>
Restriction mixture (FlhD/C):<br>
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</table><br>
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Restriction mixture (plasmid):<br>
Restriction mixture (plasmid):<br>
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DNA was extracted from gel according to protocol<br><br>
DNA was extracted from gel according to protocol<br><br>
''Results:''<br><br>
''Results:''<br><br>
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==== Ligation ====
==== Ligation ====
''Protocol:'' [https://2010.igem.org/Team:SDU-Denmark/protocols#LG1.2 LG1.2]<br><br>
''Protocol:'' [https://2010.igem.org/Team:SDU-Denmark/protocols#LG1.2 LG1.2]<br><br>
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=== Follow-up colony PCR ===
=== Follow-up colony PCR ===
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Date: 26/7<br>
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''Date:'' 26/7<br>
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Methods: Colony PCR<br>
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''Methods:'' Colony PCR<br>
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Protocol: CP1.3<br>
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''Protocol:'' CP1.3<br>
Experiment done by: Maria, LC  
Experiment done by: Maria, LC  
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<br><br>
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Notes: We made a sample for every plate from the last colony PCR. Out of the 29 samples we could only run 25 at once in the PCR machine. Plate 23 was missing, so there is no sample for it.<br><br>
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''Notes:'' We made a sample for every plate from the last colony PCR. Out of the 29 samples we could only run 25 at once in the PCR machine. Plate 23 was missing, so there is no sample for it.<br><br>
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Results: [[Image:Team-SDU-Denmark-colpcr267.jpg|600px]]<br>
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''Results:'' <br>
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[[Image:Team-SDU-Denmark-colpcr267.jpg|300px]]<br>
Lengths of PCR products: <br>
Lengths of PCR products: <br>
1200: 4, 5, 6, 9, 10, 14, 21, 26<br>
1200: 4, 5, 6, 9, 10, 14, 21, 26<br>
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Colonies 1, 15, 16 and 22 gave no result.
Colonies 1, 15, 16 and 22 gave no result.
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Analysis: Since we had so many different lengths, we will cut one from each length, specifically colony: 2, 8, 11, 13, 20, 24, 26.
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''Analysis:'' Since we had so many different lengths, we will cut one from each length, specifically colony: 2, 8, 11, 13, 20, 24, 26.
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''Results:''<br>
''Results:''<br>
gel electrophoresis:<br>
gel electrophoresis:<br>
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[[Image:Team-SDU-Denmark-Digestwithxandp.jpg|600px]]<br><br>
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[[Image:Team-SDU-Denmark-Digestwithxandp.jpg|300px]]<br><br>
''Analysis:''<br>
''Analysis:''<br>
The lanes containing digested PCR product from coloni #5, 6 and 21 have a band smear around 100-200bp,and a band at around 800bp.<br>
The lanes containing digested PCR product from coloni #5, 6 and 21 have a band smear around 100-200bp,and a band at around 800bp.<br>
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''Results:''<br>
''Results:''<br>
Gel electrophoresis:<br>
Gel electrophoresis:<br>
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[[Image:Team-SDU-Denmark-Miniprepcoloni5,6,21.jpg|600px]]<br><br>
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[[Image:Team-SDU-Denmark-Miniprepcoloni5,6,21.jpg|300px]]<br><br>
''Analysis:''<br>
''Analysis:''<br>
we have purified the plasmids.The concentrations are high but not as high as expected when the cells are boosted prior to the miniprep.<br> In order to obtain an even higher concentration transfer all 5mL ON culture to 20mL pre heated LB media. run all 25mL new culture as 1 miniprep.<br> Due to the low concentrations we need to dry down our samples before they are ready for sequentation.<br>
we have purified the plasmids.The concentrations are high but not as high as expected when the cells are boosted prior to the miniprep.<br> In order to obtain an even higher concentration transfer all 5mL ON culture to 20mL pre heated LB media. run all 25mL new culture as 1 miniprep.<br> Due to the low concentrations we need to dry down our samples before they are ready for sequentation.<br>
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<br><br>
<br><br>
Results:_Expected length at 1920 BP did not show. In fact no bands showed, except somethin like a primer smear around 100bp.<br>
Results:_Expected length at 1920 BP did not show. In fact no bands showed, except somethin like a primer smear around 100bp.<br>
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[[Image:Team-SDU-Denmark-NinaB taq colony pcr on transformants.jpg | 400px ]]
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[[Image:Team-SDU-Denmark-NinaB taq colony pcr on transformants.jpg | 300px ]]
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<br><br>
Analysis: Anealing temperature might have been set to high, as the primers were constructed to aneal at 55°. another run will be done on miniprepped plasmids, with a different program.
Analysis: Anealing temperature might have been set to high, as the primers were constructed to aneal at 55°. another run will be done on miniprepped plasmids, with a different program.
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==== Restriction digest on miniprepped pOT2 plasmids with ninaB insert using EcoRI ====
==== Restriction digest on miniprepped pOT2 plasmids with ninaB insert using EcoRI ====
Date: 27/7<br>
Date: 27/7<br>
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<br><br>
<br><br>
Results: strange things going on at different lengths, and nothing matches the expected length of about 3800BP.<br>
Results: strange things going on at different lengths, and nothing matches the expected length of about 3800BP.<br>
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[[Image:Team-SDU-Denmark-NinaB miniprep and first restriction digest-1.jpg | 800px]]
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[[Image:Team-SDU-Denmark-NinaB miniprep and first restriction digest-1.jpg |500px]]
<br>
<br>
Analysis: Another digest is needed to show that plasmid length is wrong, as it seems unlikely with comercial plasmids.<br>
Analysis: Another digest is needed to show that plasmid length is wrong, as it seems unlikely with comercial plasmids.<br>
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==== Restriction digest on miniprepped pOT2 plasmids with ninaB insert using EcoRI and XhoI ====
==== Restriction digest on miniprepped pOT2 plasmids with ninaB insert using EcoRI and XhoI ====
Date: 28/7<br>
Date: 28/7<br>
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<br><br>
results: Digest showed a band close to the 3.8kb marker expected for the full length plasmid, concurrent with only one digest working. (although an earlier digest with EcoRI alone showed a different band.) Miniprepped plasmid had also "moved up" the ladder<br>
results: Digest showed a band close to the 3.8kb marker expected for the full length plasmid, concurrent with only one digest working. (although an earlier digest with EcoRI alone showed a different band.) Miniprepped plasmid had also "moved up" the ladder<br>
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[[Image:Team-SDU-Denmark-NinaB restriction digest with eco and xho-1.jpg | 400px ]]
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[[Image:Team-SDU-Denmark-NinaB restriction digest with eco and xho-1.jpg | 300px ]]
<br><br>
<br><br>
analysis: Something fishy is up with this plasmid, and the digest. one more will be run only using XhoI.
analysis: Something fishy is up with this plasmid, and the digest. one more will be run only using XhoI.
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==== Restriction digest on miniprepped pOT2 plasmids with ninaB insert using EcoRI and XhoI ====
==== Restriction digest on miniprepped pOT2 plasmids with ninaB insert using EcoRI and XhoI ====
Date: 28/7<br>
Date: 28/7<br>

Latest revision as of 21:27, 23 October 2010