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| − | Florescence Microscopy
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| − | The following composite images were produced by superimposing a bright-field image with an YFP-filtered image.
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| − | [[File:Fluorescence_negative_control.PNG|600px|thumb|left|Figure 1, Panel A]]
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| − | Figure 1, Panel A; a composite fluorescence image – brightfield micrograph of a pSB1A3-transformed liquid cell culture (negative control) exhibiting no fluorescence as expected.
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| − | [[File:K523013 positive control.PNG|600px|thumb|left|Figure 1, Panel B]]
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| − | Figure 1, Panel B; a composite fluorescence image – brightfield micrograph of a K523013-transformed liquid cell culture exhibiting yellow fluorescence as expected.
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| − | [[File:K1352004 YFP fluorescence.PNG|600px|thumb|left|Figure 1, Panel C]]
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| − | Figure 1, Panel C; a composite fluorescence image – brightfield micrograph of a K1352004-transformed liquid cell culture exhibiting yellow fluorescence.
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| − | Confirmation of K1352004 DNA construct and the insertion of a MCS
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| − | [[File:K1352004, K523013, INP-YFP-His digest gel.png|600px|thumb|left|Figure 2]]
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| − | Figure 2; a restriction digest verification of plasmids K1352004, K523013, and “INP-YFP-His” (a previously created plasmid which identical to K1352004 except that the FLAG tag is a 6xHis tag)
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| − | For figure 2: L stands for 10,000bp – 500bp DNA marker “ladder”, and the numbers indicate the following:
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| − | 1. INP-YFP-His plasmid undigested
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| − | 2. INP-YFP-His plasmid digested with XbaI
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| − | 3. INP-YFP-His plasmid digested with XbaI and HindIII
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| − | 4. INP-YFP-His plasmid digested with XbaI and BglII
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| − | 5. K523013 plasmid undigested
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| − | 6. K523013 plasmid digested with XbaI
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| − | 7. K523013 plasmid digested with XbaI and HindIII
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| − | 8. K523013 plasmid digested with XbaI and BglII
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| − | 9. K1352004 plasmid undigested
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| − | 10. K1352004 plasmid digested with XbaI
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| − | 11. K1352004 plasmid digested with XbaI and HindIII
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| − | 12. K1352004 plasmid digested with XbaI and BglII
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| − | [[File:K1352004 standard digest.png|600px|thumb|left|Figure 3]]
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| − | Figure 3; a restriction digest verification of plasmid K1352004
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| − | For figure 3: the letters indicate the following:
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| − | L. 10,000bp – 500bp DNA marker “ladder”
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| − | N. K1352004 plasmid digested with no enzymes
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| − | E. K1352004 plasmid digested with EcoRI
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| − | X. K1352004 plasmid digested with XbaI
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| − | S. K1352004 plasmid digested with SpeI
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| − | P. K1352004 plasmid digested with PstI
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| − | EP. K1352004 plasmid digested with EcoRI and PstI
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| − | XS. K1352004 plasmid digested with XbaI and Spe1
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| − | Western Blot to confirm the presence and size of the translated INP-YFP-FLAG protein
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| − | [[File:Western blot K1352004.png|600px|thumb|left|Figure 4]]
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| − | Figure 4,
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| − | For figure 4 nitrocellulose paper, from left-to-right the lanes are:
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| − | 1. Induced (with IPTG) K1352004 lysate probed with anti-FLAG antibody #1
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| − | 2. Induced (with IPTG) K1352004 lysate probed with anti-FLAG antibody #2
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| − | 3. Induced (with IPTG) Ag43-FLAG lysate probed with anti-FLAG antibody #1
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| − | 4. Induced (with IPTG) Ag43-FLAG lysate probed with anti-FLAG antibody #2
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| − | 5. Induced (with IPTG) Ag43-FLAG lysate probed with anti-FLAG antibody #3
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| − | 6. Induced (with IPTG) Ag43-FLAG lysate probed with anti-FLAG antibody #4
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| − | 7. Protein marker “ladder”
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| − | DNA Sequencing
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| − | The recombinant plasmid was Sanger-sequenced with the following sequencing primers. “G101” is a reverse primer, the rest are forward primers.
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| − | “G101” (attaccgcctttgagtgagc)
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| − | “G100” (tgccacctgacgtctaagaa)
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| − | “35 INP-SEQ 1” (ccgattcattaatgcagctgg)
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| − | “36 INP-SEQ 2” (gaggttgctgttgccgac)
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| − | “37 INP-SEQ 3” (ggtgtggaagccgacattc)
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| − | The plasmid insert was found to be exactly as desired. Highlighted below is the MCS excerpt from the “G101” primer results (“G101” is a reverse primer which is why the sequence is in reverse complement).
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| − | [[File:MCS in INP-YFP-FLAG.JPG|600px|thumb|left|Figure 5]]
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| − | Conclusions
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| − | The construct sequence was produced exactly as designed; the full FLAG-tag – with BglII and HindIII sites – is present at the N-terminus of YFP. However, despite all this, possibly due to YFP’s C and N termini being on the same side of it, the FLAG-tag is not accessible to antibodies.
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UNIQ4d56e5fec4a93d92-partinfo-00000000-QINU
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