Team:Exeter/lab book/novpol/wk8

From 2012.igem.org

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<p><b>9.30am</p></b>
<p><b>9.30am</p></b>
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<p><a href="https://2012.igem.org/Team:Exeter/lab_book/proto/3" style="color:#1d1d1b"><u>Mini-Prep</u></a></p>
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<p><a href="http://www.fermentas.com/templates/files/tiny_mce/coa_pdf/coa_k0502.pdf" style="color:#1d1d1b"><u>Mini-Prep</u></a></p>
<p>Liquid broth transferred to new containers – leaving pipette tip behind.</p>
<p>Liquid broth transferred to new containers – leaving pipette tip behind.</p>
<p>These were then centrifuged at 3900rpg for 10 minutes.</p>
<p>These were then centrifuged at 3900rpg for 10 minutes.</p>

Revision as of 15:23, 20 September 2012

ExiGEM2012 Lab Book NovPol wk6

Showcasing Polysaccharide Production: 27th - 31st August 2012

**Tuesday 28/08/12**


9.30am

Using the NanoDrop Spectrophotometer the DNA concentrations of Becca Philp’s samples were checked for accuracy.


Table 1 Concentrations of DNA
Sample ng/μl
Cyclodextrin 1 90
Cyclodextrin 2 153
Hyaluronan Synthase (HAS) 1 66
Hyaluronan Synthase 2 92
SacB 1 106
SacB 2 94
Terminator 1 83
Terminator 2 86

10.00am

3A Assembly

Sample Conc of DNA (ng/μl) DNA required (μl) Water required (μl)
Cyc 2 150 3.3 13.5
HAS 1 330 5.5 11.3
SacB 2 100 5.0 11.8

Upstream
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

Downstream
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

Chloramphenicol 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

11.00am

Incubated for 1 hour 37°C

12.00pm

Put samples into wells of 80°C to heat denature enzymes.


1.30pm

Created gel.

Ran gel electrophoresis, 150mV for ~20 minutes.


Weighed eppendorf before and after addition of sliced gel portion containing DNA.

Re-separated DNA from gel

4.00pm

Ligation process.


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

Plasmid + Upstream + Downstream

PSBC13 + Cyc2 + Term

PSBC13 + HAS1 +Term

PSBC13 + SacB2 + Term


Ligations left at 4°C overnight.

**Wednesday 29/08/12**


2.00pm

Transformations.

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

Equilibrated water bath to 42°C.

SOC medium kept at room temperature.

One Shot TOP10 competent cells, stored at -80°C.


    1. Thawed One Shot TOP10 vials on ice for transformation.

    2. Transferred 25μl to each eppendorf to be used.
    3. Added 5μl of DNA (cyclodextrin, hyaluronan, and sacB)

2.15pm

    4. Kept the tubes on ice for 30 minutes.

2.45pm

    5. Heat shocked cells – transferred to water bath 42°C for 30 seconds exactly.
    6. Moved straight back into the ice for ~2 minutes.
    7. Aseptically added 125μl of room temperature SOC medium to each tube.

3.15pm

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

4.15pm

Span all samples at 3000G for 2 minutes.

Took 90μl off of each vial, this was then discarded.

Pipette mixed leftover contents of each eppendorf.

Using aseptic technique the rest was spread onto plates.

5.15pm

These were then put in an incubator overnight (~16+hours) at 37°C.


**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

Transferred cultures to liquid medium

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

Mini-Prep

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 2.



Table 2. 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 of 1 hour.

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

Next row contains: LADDER – SacB+Term 1,2,3,4 – HAS 1,2,3,4 – CYC 1,2,4,

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