Team:Lethbridge CCS/Notebook

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Revision as of 23:17, 6 September 2008 by Marc.slingerland (Talk | contribs)



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