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Team:Lethbridge CCS/Notebook
From 2008.igem.org
Contents |
July & August
(all team members)
- 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
13 Aug 2008
(Peter, Elizabeth, Paul, Marc, Glenda, Nathan)
- planned more specifics for project (which insert to use, etc)
19 Aug 2008
(Peter, Paul, Elizabeth, Marc, Glenda, Nathan)
- Designed primers
2 Sept 2008
(Peter, Paul, Elizabeth, Glenda, Nathan)
- Resuspended our primers (psB1A7 forward and reverse; TetR-GFP forward and reverse) to 100 μM.
- Made 10-fold dilutions of each primer
- Set up and ran PCR on psB1A7
- varied concentration of vector (1 x, 1/10 x, 1/100 x) and annealing temperatures (58°C, 62°C, 66°C), giving nine variations;
- used materials and amounts as per Phusion polymerase kit
- Cycling temperatures and times for PCR
Initial denaturation 98°C 30 sec ___
Denaturation 98°C 10 sec |
Annealing 58/62/66 °C 30 sec |--- 30 cycles
Extension 72°C 1 min ___|
Final extension 72°C 10 min
Hold 4°C hold
3 Sept 2008
(Peter, Paul, Elizabeth, Marc, Glenda, Nathan)
- PCR of pSB1A7 from yesterday worked well; approximate size was expected to be ~2500 bp, which was verified (see below)
- did PCR with TetR-GFP using same 9 samples and cycling times/temperatures as yesterday except temperature gradient was 57°C-61°C-65°
6 Sept 2008
(Peter, Paul, Marc, Glenda, Nathan)
- looked at PCR results from 3 Sept; some extra bands (see picture)
- decided to do Agarose gel purification of pSB1A7 from 2 Sept and TetR-GFP from 3 Sept
- used pooled samples as follows:
- pSB1A7: 1/100x at 62°C,1x and 1/10x at 66°C
- TetR-GFP: all three 1x samples
- used Fermentas 1kb GeneRuler; ran at 1000 V for 25 minutes
- used MinElute Gel Extraction kit to purify
6 - 13 Sept 2008
(Peter, Paul, Elizabeth, Marc, Glenda)
- spent many hours preparing and rehearsing presentation for AGEM
15 Sept 2008
(Peter, Paul, Elizabeth, Marc, Glenda)
- attended and presented at the AGEM conference in Kananaskis
24 Sept 2008
(Peter, Paul, Elizabeth, Glenda)
- prepared ligation mixture to recircularize vector
- Ligation mixture:
10x Buffer 1 μL
Vector 3 μL
Ligase 1 μL
Water 5 μL
left overnight
