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Team:Concordia/Notebook
From 2013.igem.org
Notebook
Failure for PCR amplification of Ethylene Cassette synthesis genes.
Sept, 13th
Yeast cell strain, for yeast assembly of the ethylene cassette genes, was made and thus needed its parts to assemble. The amplification of ethylene cassette genes had been previously attempted and failed. This same PCR was then attempted again on a different PCR machine and using a temperature gradient from 52 degrees to 60 degrees Celsius; the results was still unsuccessful. Upon review all reverse primers used for said genes were incorrect; this is one definite source of error!
GFP amplified in mini-prep samples
Sept, 12th
Since samples were red, and not white, presence of PompC-GFP (proof of a successful ligation) was questionable. All samples were tests for PompC using restriction digest and PCR amplification wit specific primers. Verification of these samples yielded a positive result for PompC-GFP in mini-preped samples.
Mini-Prep of PompC transformed E.coli cells.
Sept, 10th
Although all cells appeared red in color, all samples (10 in total) were mini-preped according to kit procedures.
Overlapping PCR of RBS/Plac and Fusions was unsuccessful
Sept, 9th
Our generated fusions proteins lack a Ribosome binding site (RBS) and a promoter, therefore they needed to be added in via overlapping PCR with a Plac template and specific primers. This PCR was attempted in two different manners; one was done in a temperature gradient PCR from 58 degrees to 68 degrees Celsius, and the other was conducted using trouble shooting techniques (such as DNA dilutions and taq polymerase). Both were unsuccessful and no product was obtained.
Overlapping PCR of RBS/Plac and Fusions was unsuccessful
Sept, 10th
Our generated fusions proteins lack a Ribosome binding site (RBS) and a promoter, therefore they needed to be added in via overlapping PCR with a Plac template and specific primers. This PCR was attempted in two different manners; one was done in a temperature gradient PCR from 58 degrees to 68 degrees Celsius, and the other was conducted using trouble shooting techniques (such as DNA dilutions and taq polymerase). Both were unsuccessful and no product was obtained.
Amplification of RhlI, LuxR, and LuxI. Purification of all Fusion proteins and Plac.
Sept, 3rd
Continuing amplification of interface components yielded successful amounts of RhlI in several experiments. However, LuxR and LuxI were not amplifying out under normal conditions; therefore several troubleshooting parameters were attempted. Out of four parameters altered, only the increased Magnesium content yielded results. The bands obtained of each product were weak at best. More troubleshooting needs to be attempted. The previously generated fusions (DOM,CON and LIT) were purified, via ethanol purification, as well as Plac.
Ethanol Purification of ETR1, PLac, and PompC-GFP
Aug, 22nd
ETR1, PLac and PompC-GFP were Ethanol Purified.
Amplification of ETR1, LIT fusion, PLac, and Pomc-GFP.
Aug, 21st
Troubleshooting PCR amplification of LIT fusion and ETR1 using a temperature gradient (from 58 degrees C to 62 degrees C). Normal PCR amplification of PLac and PompC-GFP. All products are at the correct size!
Amplification/Ethanol Purification of TetR and RhlR. Ethanol Purification of PompC-GFP
Aug, 13th
PCR amplification of interface genes was commenced; RhlR was successfully amplified and purified. RhlR was quantified to be 360 ng/ul. Gas clock component 'TetR' was also successfully amplified, and later purified. TetR quantification yield was 216 ng/ul. Additionally, PompC-GFP was successfully ethanol purified and quantified to be approximately 270 ng/ul.
Phire Discontinued
Aug, 12th
The Phire DNA polymerase sample we used was obtained from the Dr.Martin lab. Since it was only a trial sample, more Phire DNA polymerase needed to be ordered. We absolutely wanted to order Phire DNA polymerase since it had proven to be successful previously (Aug 01). But when we attempted to order it we figured out that the original Phire, and its second generation 'Phire Hot start Green polymerase' had been discontinued.
Ethanol purification of EnvZ, and Fusions DOM/CON
Aug, 7th
Amplification of ETR1 and LIT Fusion were attempted again but were unsuccessful. On the other hand, the Ethanol Purification of EnvZ, and Fusions DOM and CON were carried out successfully! Concentrations, via Gel Quantification, are estimated as; 630 ng/ul for EnvZ, 450ng/ul for DOM fusion, and 105 ng/ul for CON fusion.
Amplified EnvZ, but No LIT fusion
Aug, 6th
We needed ETR1 and EnvZ for Nested Deletion and an amplification of LIT fusion, so an amplification PCR was conducted. For our Nested Deletions; EnvZ was successfully Amplified from E.coli genomic DNA, but ETR1 was not amplified from A. thaliana cDNA. Sadly, our LIT fusion amplification, using DNA template from the successful overlapping PCR, was not successful.
Low Purification Yield; Fusions
Aug, 2nd
Our Fusions that were generated using 'Phire DNA polymerase' were Gel Purified and visualized using Gel Electrophoresis. What we saw was not good news, Fusions LIT and CON were not concentrated enough. DOM, and CON were usable but LIT was not. LIT fusion has to be amplified.
Generated Fusions
Aug, 1st
Overlapping PCR; Fusion Generation, from E.coli EnvZ and A. thaliana ETR1, had been giving us problems so we applied a troubleshooting technique using various DNA polymerases. Out of the four polymerases used, only one gave us our Fusions (DOM,CON,LIT) at the expected sizes. 'Phire DNA polymerase' is amazing!
