Team:Lethbridge CCS/Notebook/September

From 2008.igem.org

For earlier work, see July & August Notebook, or go back to Main CCS Notebook.

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 by agarose gel

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 on agarose gel
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, Nathan)

prepared ligation mixture to recircularize vector

- Ligation mixture:
    10x Buffer     1 μL
    Vector         3 μL
    Ligase         1 μL
    Water          5 μL
  left overnight

27 Sept 2008

(Peter, Paul, Elizabeth, Marc, Glenda, Nathan)

cultures grew nicely, but need to do further tests to see if what grew was what we were looking for
spent some time discussing which buffers, temperatures, times, etc. to use with T4 endonuclease for testing the LIC method and the Klenow fragment of EcoR1 endonuclease for trying the SLIC method
decided on using Fermentas buffers for T4 and NEB buffer 2 for Klenow; decided we would use same conditions for both: 20 minutes at 37 °C, followed by 10 minutes at 65 °C.

29 Sept 2008

(Paul, Elizabeth, Marc, Glenda, Nathan)

Nathan had picked four colonies and grew up in 5 mL culture overnight

Glycerol stocks: for each culture, combined 500 μL sterilized glycerol with 500 μL cell culture & stored in HJ's -80°C freezer

Plasmid prep:
- spun down 1.4 mL of culture media to pellet cells (1 minute, 14000 rpm)
- followed directions for QIAprep Spin Miniprep Kit to purify plasmid DNA

DNA Quantification: analyzed 25-fold dilution of each purified pDNA sample using Ultraspec 3300pro UV/visible spectrophotometer (1s integration)

Sample #	A @ 260 nm	A @ 280 nm	DNA Concentration (μg/mL)
1	0.073	0.045	81
2	0.084	0.054	90
3	0.061	0.039	65
4	0.101	0.066	100

purified pDNA was stored in freezer for later use

30 Sept 2008

(Paul, Elizabeth, Glenda, Nathan)

did gel electrophoresis on 4 colonies chosen from smear plate of recircularized plasmid; all same length
treated our vector and insert with polymerase (each were done with both T4 and Klenow)

 - T4 treatment: components and amounts
    dCTP           3.33 μL                               dGTP            3.33 μL
    T4 Buffer         2 μL                               T4 Buffer          2 μL
    T4              0.4 μL (2 units)                      T4              0.4 μL
    Vector          2.5 μL                                Insert          2.5 μL
    Water          1.77 μL                               Water           1.77 μL

 - Klenow treatment: components and amounts
    NEBuffer2      1 μL                                  NEBuffer2       1 μL
    Klenow         2 μL                                  Klenow          2 μL
    Vector         1 μL                                  Insert          2.5 μL
    Water          6 μL                                  Water           4.5 μL
  (Smaller amount of vector used as we did not have any more; probably should have used larger volume as 
   polymerase volume should be 10% of total volume)

we then did a PCR purification using the Qiagen MinElute Purification kit, followed by quantification on all 4 of the samples prepared above

- at 50x dilution, T4-GFP gave ratio of 1.6 and concentration of 92 ng/μL, while T4-vector gave ratio of 1 
  and concentration of 42 ng/μL and both Klenow samples gave basically no results
- we then repeated the T4-vector at 25x dilution and obtained a ratio of 1.05 and concentration of 54 ng/μL; 
  we also repeated the Klenow samples at 16x dilution, but results were still inconclusive
- we decided we would mix the remaining Klenow samples together and run a transformation
-NOTE: water was used as blank, rather than EB buffer as ought to have been used

we also did an EcoRV digestion and then ran a gel

- components and amounts (for each of 4 colonies grown up previously)
    DNA      5 μL                             
    Buffer   1 μL                                  
    EcoRV    1 μL
    Water    3 μL

For later work, see October Notebook.

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