Team:EPF-Lausanne/Notebook/July2011

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This time the purification will be performed not from the gel but directly from PCR with a commercial kit.
This time the purification will be performed not from the gel but directly from PCR with a commercial kit.
[[File:EPFL2011_pSB3K1_PCR_260711.jpg|thumb|right|pSB3K1 successfully amplified by PCR]]
[[File:EPFL2011_pSB3K1_PCR_260711.jpg|thumb|right|pSB3K1 successfully amplified by PCR]]
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Alessandro send the Vincent's plasmid (J6001-Ptet-RFP) to be sequenced (as the primer arrived).
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In the mean time, Doug ran an identical gradient PCR with Matt's HiFiPLUS enzyme, changing the buffer concentration to 10 ul per 50 ul and lengthening the extension step to 2'30", as recommended by the datasheet.
In the mean time, Doug ran an identical gradient PCR with Matt's HiFiPLUS enzyme, changing the buffer concentration to 10 ul per 50 ul and lengthening the extension step to 2'30", as recommended by the datasheet.
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== Wednesday, 27 July 2011 ==
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Alessandro purified the pSB3K1 backbone (to use for Gibson assembly and make the Pconst-TetR plasmid) from the PCR of the day before getting 60.6 ng/uL.
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The sequencing reaction failed and checking the conditions of primers and template with NanoDrop, it seems like reverse primer has no DNA (but this doesn't explain why the reaction failed also with the forward primer); there's also a weird thing to annotate: checking the template on NanoDrop we always have an error of calibration.
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Alessandro made new sample to send again sequencing.
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We '''FINALLY HAVE A WORKING PCR!!!!1111one''''.
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[[File:EPFL-2011-07-27_Gibson_gradient_PCR_hifiPLUS.jpg|thumb|right|Backbone and Lysis fragments for the reporter plasmids, PCR'd with HifiPLUS. Expected product lengths: 2.4 kb backbone, 1.9 kb Lysis cassette.]]
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More seriously, a gel was run on yesterday's gradient PCR products --- the PCR was ran using the High Fidelity PLUS enzyme, on the J61002-pTet backbone and pLac-T4Lysis fragments, for the Gibson assembly of the reporter plasmids. For both fragments, high quantities of specific product were yielded, though the backbone also contains large amounts of non-specific product.
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For the backbone, lower annealing temperatures yield more product, as expected. The non-specific product seems relatively unaffected by annealing temperature (or it may decrease slightly at lower temperatures). The unspecific product present in large amounts is of similar length to the RFP gene, which is also present on the backbone template --- but determining its origin is unnecessary: it doesn't contain an antibiotic resistance gene, so plasmids assembled from it would not allow culture growth in the amplification step.
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The T4 Lysis cassette fragment is a little too small. It could be due to GelRed affecting migration speed (according to Alina, it happens with large amounts of DNA), or it could indeed be an undesired product of shorter length. Please note the product quantities are barely affected by temperature (to the extent we can eyeball from the gel), whereas the expected primer melting temperatures are between 52° and 57°C, so we do not expect any product with 65°C annealing steps.
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The assembly step was started. Transformation can be started tomorrow morning.
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'''For the following PCRs''': Use Roche High Fidelity PLUS enzyme, and adapt any protocols to the appropriate specifications by Roche. Also, use either bottled water or the newer jar with fresher water (the one with autoclave tape on the lid).
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== Thursday, 28 July 2011 ==
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The sequencing reaction this time went good therefore the plasmid is definitely the one expected.
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Alessandro transformed and plated the plasmid to perform the day after the colony PCR just to optimize the protocol (this will be our positive control) for this technique that will be used to screen the gibson assembly.
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Since seems to be some debris in the SOC medium bottle, we also plated it (under the same conditions of the cells) to test if something grows. Alessandro also tested the OD of the medium after 1 hour of incubation and no significant difference was found.
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Douglas transformed and plated the Gibson-assembled reporter plasmids at the same time as Alessandro's plasmids. He later transformed it in a different ''E. Coli'' colony that over-expressed LacI, and therefore has a reduced probability of expressing the holin (from the lysis cassette) before they synthesise enough LacI to repress it.
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Douglas also repeated the '''mutation PCR''' using the new High Fidelity PLUS enzymes. In a bold and dareful move, he programmed the thermal cycler to carry out both a touchdown PCR AND progressively extended elongation steps (during the second set of cycles, those at the floor annealing temperature, the elongation step is extended of five seconds every cycle).
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== Friday, 29 July 2011 ==
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Douglas and Alessandro's transformed cells didn't grow (maybe problems with competent cells?).
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Alessandro transformed the cells again (with the J61002-Ptet-RFP Gibson plasmid) but this time he trasformed also the sample labeled Col1-RFP and Col4-RFP (supposing that these are the same as J61002-Ptet-RFP Gibson plasmid).
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Alessandro transformed the Gibson assembly made by Douglas on Lilia's (or Alina's?) competent cells and also on Henrike's competent cells. Due to an error only the lysis cassette has been transformed. On Tuesday the Gibson assembly has to be repeated since we ran out of the previous one.
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The Mutation PCR yielded no product, probably because the primers were too diluted. It has been repeated with a higher primer concentration.
{{:Team:EPF-Lausanne/Templates/Footer}}
{{:Team:EPF-Lausanne/Templates/Footer}}

Latest revision as of 16:18, 2 August 2011

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