Team:Exeter/lab book/novpol/wk7

From 2012.igem.org

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<p>All samples were spun down.</p>
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<b><p>2.45pm</p></b>
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<p>Incubated all tubes. For digestion period.</p>
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<b><p>3.55pm</p></b>
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<p>Samples out of incubator. 4μl of Loading Buffer added to each. These were then transferred to individual wells. </p>
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<p>LADDDER – HAS+Term 1,2,3,4 – CYC+Term 1,2,3,4 WbiP 2, 3, 4 </p>
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<p>New line contains: LADDER – SacB+Term 1,2,3,4 – HAS 1,2,3,4 – CYC 1,2,4,</p>
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Revision as of 16:44, 2 September 2012

ExiGEM2012 Lab Book NovPol wk7

Showcasing Polysaccharide Production: 27th - 31st August 2012

Thursday 30/08/12


9.30am

Checked plates. Colonies have formed on all.

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

4.30pm

Made up liquid broth using plates:

    * pSB1C3 cyclodextrin
    * pSB1C3 hyaluronan synthase
    * pSB1C3 cyclodextrin + terminator
    * pSB1C3 hyaluronan synthase + terminator
    * pSB1C3 sacB + terminator

4x made per plate – aseptic technique

Used 10ml broth

10μl chloramphenicol

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

5.30pm

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

Friday 31/08/12


9.30am

Liquid broth transferred to new containers – leaving pipette tip behind.

These were then centrifuged at 3900rpg for 10 minutes.

    1. supernatant discarded leaving pellet at the base.

    2. re-suspended pellet in 250μl re-suspension buffer, used up/down pipette mixing. Moved into eppendorf tubes.
    3. added 250μl lysis buffer, mixed each by turning (upside down and back).
    4. added 350μl neutralisation buffer.
    5. centrifuged at full speed (13k) for 5 minutes. White build up had formed itself in the centre of the vials, centrifuged again for 5 minutes. Slight change, clear fluid became accessible.
    6. clear fluid transferred to flow through tubes.
    7. centrifuged for 1 minute --> flow through fluid discarded --> 500μl of wash solution added.
    8. centrifuged for 1 minute --> flow through fluid discarded --> 500μl of wash solution added again.
    9. centrifuged for 1 minute --> flow through fluid discarded --> centrifuged for 1 minute.
    10. transferred column to clean eppendorf.
    11. added 50μl clean water --> left for 2 minutes --> centrifuged for 2 minutes, kept vial, discarded column.


12.30am

Samples were then placed on the NanoDrop machine to get the concentration of DNA.

Using this and the measurements from the previous digestion, seen below in table 1.



Table 1. Digest Measurements
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

The amount of DNA and water required for all samples could be calculated.


Sample Conc of DNA DNA required Water required
CYCLODEXTRIN ng/μl μl μl
1 40.7 2.29 4.51
2 55.8 8.93 7.90
3 41.1 12.2 4.6
4 947.5 0.53 16.3
HYALURONAN SYNTHASE ng/μl μl μl
1 194.1 2.58 14.22
2 106.6 4.69 12.11
3 40.0 12.5 4.3
4 94155.4 3.22 13.58
CYCLODEXTRIN +TERMINATOR ng/μl μl μl
1 100.4 5.0 11.8
2 142.8 3.5 13.3
3 16.3 30.7 1.0
4 110.8 4.5 12.3
HYALURONAN SYNTHASE + TERMINATOR ng/μl μl μl
1 104.6 4.8 12
2 211.7 2.36 14.4
3 55.5 9.0 7.8
4 70.4 7.1 9.7
SacB + TERMINATOR ng/μl μl μl
1 119.3 4.2 12.6
2 22.5 22.0 1.0
3 84.0 6.0 10.8
4 67.9 7.4 9.4

All samples were spun down.

2.45pm

Incubated all tubes. For digestion period.

3.55pm

Samples out of incubator. 4μl of Loading Buffer added to each. These were then transferred to individual wells.

LADDDER – HAS+Term 1,2,3,4 – CYC+Term 1,2,3,4 WbiP 2, 3, 4

New line contains: LADDER – SacB+Term 1,2,3,4 – HAS 1,2,3,4 – CYC 1,2,4,

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