Team:UCSC/Results
Results
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YEAST MEDIATED CLONING
We concluded the successful removal of loxP from pXRL2 via site directed mutagenesis. The results of our colony PCR suggested that the product of the reaction was slightly smaller than the original plasmid. However, the positive control had a strange band higher than we expected. We sequenced to confirm our success and found that the product was missing the desired region in an alignment with the original plasmid. We also conducted attempts of yeast mediated cloning with successful growth in leucine deficient media. This result would suggest that we had a successful transformation and possible assembly of our gene cassette on to the plasmid backbone.
We conducted a colony PCR to confirm the successful site directed mutagenesis of loxP from pXRL2. We used a pair of primers: cloxP F and cloxP R to amplify the region that we removed and ran the samples on a gel. A successful trial would be a band 300 bp or lower since the fragment including the lox site would be 343 bp. Our results were puzzling due to the band being higher than 300 bp but we thought that this had occurred because the sample ran slower than the other lanes evident in the arc of the dye. We conducted another colony PCR with monoclonal colonies. Strangely, our positive control appeared to be much larger than the other samples but we concluded that it was due to issues with gel electrophoresis. We resolved to use the samples with the lowest bands or smallest size which were 5C, 5D, 21B and 23A. Upon further investigation by sequencing, we concluded that we had succeeded due to the region being missing in an alignment with the original plasmid. However, in sample A, more bases were deleted than expected so we did not use it because of risk of a frameshift. We, then relinearized the four sequenced samples for gene assembly via yeast mediated cloning. The results of this suggested a success since the bands resulting from gel electrophoresis were at the expected 8-10 kb.
GIBSON
pUC19 and PXRL2 Linearization
We linearized plasmids pUC19 and pXRL2 to prepare for Gibson Assembly and to cut out the first loxP site in preparation for YMC. The first few times we tried, there were no bands of any length. This occurred for 5 rounds of PCR until we discovered there was an issue with our Q5 MasterMix, and resolved it. Once we fixed that, we got successful linearization results. \figref{PlGel} shows our gel with plasmid pUC19 linearization in Lane 1, with a band a little higher, but around the expected size of 2.7kb, and plasmid pXRL2 linearization in Lane 2, with a band around the expected size of 7kb.
We conducted a colony PCR to confirm the successful site directed mutagenesis of loxP from pXRL2. We used a pair of primers: cloxP F and cloxP R to amplify the region that we removed and ran the samples on a gel. A successful trial would be a band 300 bp or lower since the fragment including the lox site would be 343 bp. Our results were puzzling due to the band being higher than 300 bp but we thought that this had occurred because the sample ran slower than the other lanes evident in the arc of the dye. We conducted another colony PCR with monoclonal colonies. Strangely, our positive control appeared to be much larger than the other samples but we concluded that it was due to issues with gel electrophoresis. We resolved to use the samples with the lowest bands or smallest size which were 5C, 5D, 21B and 23A. Upon further investigation by sequencing, we concluded that we had succeeded due to the region being missing in an alignment with the original plasmid. However, in sample A, more bases were deleted than expected so we did not use it because of risk of a frameshift.