Team:Exeter/lab book/novpol/wk9

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Our own competent cells were removed from freezer, -80°C.
Our own competent cells were removed from freezer, -80°C.
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<ul><b><i>1.</i></b> 5μl of DNA from each sample, Tetr_RBS + Cyc _Term & Has_Term and OmpA + SacB_Term added to 50μl of competent cells. The reason for using the entire quantity of competent cells was because they were believed to less efficient than TOP10. </ul>  
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<ul><b><i>1.</i></b> 5μl of DNA from each sample, Tetr_RBS + Cyc _Term & Has_Term and OmpA + SacB_Term added to 50μl of competent cells. The reason for using the entire quantity of competent cells was because they were believed to be less efficient than TOP10. </ul>  
<ul><b><i>2.</i></b> Kept on ice for 30 minutes. Whilst heat bath equilibrates to 42°C.</ul>
<ul><b><i>2.</i></b> Kept on ice for 30 minutes. Whilst heat bath equilibrates to 42°C.</ul>
<p><b>3.20pm</p></b>
<p><b>3.20pm</p></b>

Revision as of 10:37, 13 September 2012

ExiGEM2012 Lab Book NovPol wk6

Showcasing Polysaccharide Production: 3rd - 7th September 2012

**Monday 03/09/12**


9.00am

Transformations using Gene_Terminator.

    * Tetr_RBS + Cyc_Term
    * Tetr_RBS + HAS_Term
    * ompA + sacB_Term
Sample Conc of DNA (ng/μl) DNA required (μl) Water required (μl)
Cyc_Term 2 142.8 3.5 13.3
HAS_Term 1 104.6 4.8 12.0
SacB_Term 1 119.3 4.2 12.6
Kanamycin Plasmid 112.8 4.43 12.37
Tetr_RBS 42.2 11.85 4.95
OmpA 70.2 7.12 9.68

Upstream
DNA 500ng
BUFFER (10x) 2μl
BSA (100x) 0.2μl
Water up to 20μl total V
ENZYME 1 - XbaI 0.5μL
ENZYME 2 - PstI 0.5μL

Downstream
DNA 500ng
BUFFER (10x) 2μl
BSA (100x) 0.2μl
Water up to 20μl total V
ENZYME 1 - ECORI 0.5μL
ENZYME 2 - SpeI 0.5μL

Kanamycin Plasmid
DNA 500ng
BUFFER (10x) 2μl
BSA (100x) 0.2μl
Water up to 20μl total V
ENZYME 1 - ECORI 0.5μL
ENZYME 2 - PstI 0.5μL

11.20am

All in the incubator for digestion period.

12.40pm

Heat denatured enzymes at 80°C for 20 minutes.

1.00pm

Ligation process


Ligation
Backbone 2μl
Upstream 2μl
Downstream 2μl
DNA ligase buffer 1μl
DNA ligase enzyme 0.5μL
Water 2.5μL

Plasmid + Upstream + Downstream

Plasmid + Tetr_RBS + Cyc_Term

Plasmid + Tetr_RBS + HAS_Term

Plasmid + OmpA + SacB_Term

2.50pm

Transformations

Our own competent cells were removed from freezer, -80°C.
    1. 5μl of DNA from each sample, Tetr_RBS + Cyc _Term & Has_Term and OmpA + SacB_Term added to 50μl of competent cells. The reason for using the entire quantity of competent cells was because they were believed to be less efficient than TOP10.
    2. Kept on ice for 30 minutes. Whilst heat bath equilibrates to 42°C.

3.20pm

    3. Heat shocked cells – transferred to water bath 42°C for 30 seconds exactly.
    4. Moved straight back into ice for ~2 minutes.
    5. Aseptically added 700μl of room temperature LG medium to each eppendorf.

3.30pm

    6. Incubated all samples in the shaker at 37°C, 250 rpm for 1 hour.

    7. Span samples at 3000G for 5 minutes.

      Plates labelled:

      Tetr_RBS + cyc_Term

      Tetr_RBS + HAS_Term

      OmpA + SacB_Term

      Aseptically discarded 600μl solution from each eppendorf. Pipette mixed remaining and spread over plates.

      5.05pm

      Plates in incubator, 37°C overnight.


      **Tuesday 04/09/12**


      9.00am

      Checked plates. Colonies have formed on all. Our own competent cells appear to be more efficient than first expected, less should be spread on future plates to make single colonies easier to pick.

      These were all placed in the fridge, 4°C.

      3.25pm

      Plates out of fridge, read to make liquid broth.

      4x made per plate – aseptic technique

      Used 10ml broth

      10μl Kanamycin

      Scraped off single colony using pipette tip which was then ejected into liquid broth.

      Tetr_RBS + HAS_Term and OmpA + SacB_Term had red and white colonies on the plates. Only the white colonies were selected.

      4.20pm

      Put into horizontal shaker set at 37°C, 220rpm – left overnight.

      **Wednesday 05/09/12**


      9.00am

      Liquid broth aseptically transferred to new falcon tubes – leaving pipette tip behind.

      10.00am

      These were then centrifuged at 3900rpg for 10 minutes, 4°C.

      The same protocol was followed as in the previous week for mini preps. However 40μl of clean water was added, instead of the previous 50μl, to boost the concentration of DNA in each sample.

      1.30pm

      Sample Conc of DNA DNA required Water required
      Tetr_RBS + Cyc_Term ng/μl μl μl
      1 136.8 3.65 13.15
      2 154.4 3.24 13.56
      3 126.5 3.95 12.85
      4 114.8 4.36 12.44
      Tetr_RBS + HAS_Term ng/μl μl μl
      1 68.4 7.31 9.49
      2 65.3 7.66 9.14
      3 100.6 4.97 11.83
      4 92.8 5.39 11.41
      OmpA + SacB_Term ng/μl μl μl
      1 123.6 4.05 12.75
      2 116.7 4.28 12.52
      3 250.1 2.00 14.8
      4 95.7 5.22 11.58

      2.45pm

      Currently no enzymes, all DNA into freezer.

      4.30pm

      Streak plates made from the single colonies that appeared to have worked. Colonies that form should therefore also work.

        HAS_Term 1 & 4
        Cyc_Term 1, 2 & 4
        SacB_Term 1, 3 & 4
        HAS 1, 2, 3 & 4
        Cyc 1 & 2

      All into incubator at 37°C overnight.



      **Thursday 06/09/12**


      3.30pm

      Digestion


      Digestion
      DNA 500ng
      BUFFER (10x) 2μl
      BSA (100x) 0.2μl
      Water up to 20μl total V
      ENZYME 1 - ECORI 0.5μL
      ENZYME 2 - PstI 0.5μL

      Into incubator for overnight digestion.



      **Friday 07/09/12**


      11.00am

      Gel electrophoresis run - insert image here



      Sequences confirmed for:

        Cyclodextrin_Terminator
        HAS_Terminator
        SacB_Terminator

      Single genes confirmed for:

        Cyclodextrin
        HAS

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