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| <!-- {{#calendar: title=Lethbridge_CCS |year=2008 | month=05}} --> | | <!-- {{#calendar: title=Lethbridge_CCS |year=2008 | month=05}} --> |
| + | [[Image:Lethbridge_CCS_logo.png|Logo|100px]] [[Team:Lethbridge_CCS|Team Home]] |
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| + | <!-- === July & August === --> |
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| + | '''[[Team:Lethbridge_CCS/Notebook/July & August| July & August Notebook]]''' |
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- | === July & August ===
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- | (all team members)
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- | * as time permitted, we spent time with the U of L team to watch, ask questions about, and try techniques we would be using once we got started on our own project
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- | === 13 Aug 2008 ===
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- | (Peter, Elizabeth, Paul, Marc, Glenda, Nathan)
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- | * planned more specifics for project (which insert to use, etc)
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- | === 19 Aug 2008 ===
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- | (Peter, Paul, Elizabeth, Marc, Glenda, Nathan)
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- | * Designed primers
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- | === 2 Sept 2008 ===
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- | (Peter, Paul, Elizabeth, Glenda, Nathan)
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- | * Resuspended our primers (psB1A7 forward and reverse; TetR-GFP forward and reverse) to 100 μM.
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- | * Made 10-fold dilutions of each primer
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- | * Set up and ran PCR on psB1A7
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- | ** varied concentration of vector (1 x, 1/10 x, 1/100 x) and annealing temperatures (58°C, 62°C, 66°C), giving nine variations;
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- | ** used materials and amounts as per Phusion polymerase kit
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- | - Cycling temperatures and times for PCR
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- | Initial denaturation 98°C 30 sec ___
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- | Denaturation 98°C 10 sec |
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- | Annealing 58/62/66 °C 30 sec |--- 30 cycles
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- | Extension 72°C 1 min ___|
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- | Final extension 72°C 10 min
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- | Hold 4°C hold
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- | === 3 Sept 2008 ===
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- | (Peter, Paul, Elizabeth, Marc, Glenda, Nathan)
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- | * PCR of pSB1A7 from yesterday worked well; approximate size was expected to be ~2500 bp, which was verified (see below)
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- | ** [[Image:PCRpSB1A7]]
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- | * did PCR with TetR-GFP using same 9 samples and cycling times/temperatures as yesterday except temperature gradient was 57°C-61°C-65°
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- | === 6 Sept 2008 ===
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- | (Peter, Paul, Marc, Glenda, Nathan)
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- | * looked at PCR results from 3 Sept; some extra bands (see picture)
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- | ** [[Image:PCRTetR-GFP]]
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- | * decided to do Agarose gel purification of pSB1A7 from 2 Sept and TetR-GFP from 3 Sept
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- | * used pooled samples as follows:
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- | ** pSB1A7: 1/100x at 62°C,1x and 1/10x at 66°C
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- | ** TetR-GFP: all three 1x samples
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- | * used Fermentas 1kb GeneRuler; ran at 1000 V for 25 minutes
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- | * used MinElute Gel Extraction kit to purify
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| + | '''[[Team:Lethbridge_CCS/Notebook/September|September Notebook]]''' |
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| + | '''[[Team:Lethbridge_CCS/Notebook/October|October Notebook]]''' |
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| + | <div style="display:block; text-align:center; border:2px solid #6C5; width:500px; margin:auto; margin-top:3em; padding:5px;"> |
| + | <p> [[Team:Lethbridge_CCS | Main Page]] | [[Team:Lethbridge_CCS/Team | Team ]] | [[Team:Lethbridge_CCS/Project | Project ]] | [[Team:Lethbridge_CCS/Notebook | Lab Notebook]] </p> |
| + | </div> |
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| <!-- ==Notebook== | | <!-- ==Notebook== |