Team:Imperial College London/Lab Diaries/XylE team
From 2010.igem.org
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+ | <title>Imperial iGEM 2010</title> | ||
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+ | <p id="popshow">Follow our progress: Click me!</p> | ||
+ | <div id="popcon"> | ||
+ | <ul id="poplist"> | ||
+ | <li><a href="#Week_6" class="popt">Week 6</a></li> | ||
+ | <li><a href="#Week_7" class="popt">Week 7</a></li> | ||
+ | <li><a href="#Week_8" class="popt">Week 8</a></li> | ||
+ | <li><a href="#Week_9" class="popt">Week 9</a></li> | ||
+ | <li><a href="#Lab_Diary" class="popt">Lab Diary</a></li> | ||
+ | <li><a href="#Output_Photo_Gallery" class="popt">Output Photo Gallery</a></li> | ||
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[[Image:wk8.jpg|thumb|800px|16.8.10-20.8.10]] | [[Image:wk8.jpg|thumb|800px|16.8.10-20.8.10]] | ||
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+ | ===Monday, 23-Aug=== | ||
+ | *set up overnight cultures for midi-prep | ||
+ | *gel separation of linker-XylE-Spe DNA <font color = red>'''FAILED'''</font> | ||
+ | *PCR reaction for extension of His-GFP-Flag | ||
+ | *Catechol assay on E.coli transformed with overnight ligated J23101, XylE and pSB1C3. <font color = red>'''FAILED'''</font> | ||
+ | |||
+ | ===Tuesday, 24th-Aug=== | ||
+ | |||
+ | *midi-prepped registry obtained J23101. A yield of 130ng/ul of promoter was obtained. The promoter is in a biobrick vector called J62001. The promoter is upstream of RFP gene. | ||
+ | *the vector carrying the promoter was digested with SpeI and PstI, while XylE gene was digested with XbaI and PstI. | ||
+ | *the promoter and the XylE gene were gel purified. | ||
+ | *a reaction between the promoter(still on vector) and XylE was set on for overnight ligation | ||
+ | *PCR purification of GFP2 -> GFP construct ready for full fusion protein. | ||
+ | *Gel purification of XylE lost DNA along the way. Thus PCR XylE1 with gradient for temperature scanning (taq): PCR round 1 included 62C and only rev primer to create pool of successful extensions with with rev primer (60-62). PCR round 2 with Fwd primer and temperature scale (68-68, 72-74). | ||
+ | |||
+ | ===Wednesday, 25th-Aug=== | ||
+ | |||
+ | Performed gel analysis on the purified XylE and J23101 to obtain ratios for ligation. First gel was scrapped as it produced appauling(explanation for Nick:really bad) results, 2nd gel run was successful. | ||
+ | *Performed a ligation reaction between the vector containing J23101, and XylE(one on bench and one overnight one). | ||
+ | *Transformation of the new plasmid into competent E.coli. Successfully transformed colonies can be selected for by loss of RFP expression. | ||
+ | * XylE-1 PCR with temperature cascade. Gel analysis and purification. | ||
+ | |||
+ | ===Thursday, 26th-Aug=== | ||
+ | |||
+ | *white colonies from the promoter-XylE transformed E.coli were picked and transferred to new amp plates. One is the replica plate and the other is the catechol assay plate. | ||
+ | * XylE-1, two rounds of PCR/purification were run to obtain a sufficiently clear band. An additional PCR run for XylE-1 was discarded afterwards. | ||
+ | |||
+ | ===Friday, 27th-Aug=== | ||
+ | |||
+ | *Catechol assay performed on promoter-XylE transformed E.coli. <font color = green>'''SUCCESSFULL'''</font> | ||
+ | [[Image:Catechol Assay before.jpg|thumb|left|Plate before adding catechol assay]] [[Image:Catechol assay after (27-8).jpg|thumb|center|After addition of catechol colonies turn yellow-orange in seconds!!]] | ||
+ | * XylE-2 PCR and gel-purification cycles (2x) to obtain clear band. XylE-2 is now ready for assembly of the GFP-XylE fusion protein. | ||
+ | |||
+ | ===Saturday, 28th-Aug=== | ||
+ | |||
+ | * Preparing the annealing step between the GFP-2 and XylE-2 constructs, we discovered sequence dissimilarities in the TEV-cleavable regions which we planned to use for the annealing step. Nevertheless a PCR was run with appropriate conditions (allowing for a minimal amount of unspecific annealing). | ||
+ | |||
+ | ===Sunday, 29th-Aug=== | ||
+ | |||
+ | * Gel analysis of the attempted annealing reaction of GFP-2 XylE-2 showed unsufficiently clear bands for gel-purification. A new reaction is being prepared: 10 rounds of annealing PCR, followed by addition of primers (5' primer for GFP-2 and 3' primer for XylE-2) in order to introduce an amplification step in the reaction. --- Following Kirstins advice, we are discarding this reaction and wait for the arrival of new primers for XylE-2 (5' + TEV) and GFP-2 (3' +TEV). |
Revision as of 09:27, 21 October 2010
Lab Diaries | Overview | Surface Protein Team | XylE Team | Vectors Team | Modelling Team |
Here are the technical diaries for our project. We've split them up into three lab teams and the modelling team. We think it's really important that absolutely anyone can find out what we've been doing. For a really detailed look at what we did, and when, you've come to the right place! |
Team XylE |
Follow our progress: Click me!
XylE team Lab Objectives |
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Lab notes and schedule |
Week 6Thursday, 12-Aug-2010
we constructed the standard E.coli promoter J23101 with sticky ends. These ends are complementary to restriction sites made by EcoRI and SpeI enzyme. This promoter will be later used in 3A assemply to construct a promoter-RBS-XylE design in a psB1C3 vector. E.coli will be transformed with this final construct plasmid to assess XylE activity and characterization. It will also be one of the submitted biobricks.
these cultures are going to be used tomorrow for mini-prepping. Miniprep will allow us to isolate E.coli's plasmid DNA(which contains the XylE gene). Friday, 13-Aug-2010
Mini-prep is usually used to confirm that our gene of interest has not been changed in any way, as the isolated plasnid id sent for sequencing. However, since XylE was taken from the registry, we assume that it is fine and no sequencing is required. The mini-prep will later be used for the midi-prep (that gives out higher yeilds of DNA needed for cloning).
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Week 7Monday, 16-Aug-2010
Tuesday, 17-Aug-2010
Thursday, 19-Aug-2010
Friday, 20-Aug-2010The J23101 gene in a biobrick vector containg RFP gene
Week 8Monday, 23-Aug
Tuesday, 24th-Aug
Wednesday, 25th-AugPerformed gel analysis on the purified XylE and J23101 to obtain ratios for ligation. First gel was scrapped as it produced appauling(explanation for Nick:really bad) results, 2nd gel run was successful.
Thursday, 26th-Aug
Friday, 27th-Aug
Saturday, 28th-Aug
Sunday, 29th-Aug
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