Team:Exeter/lab book/novpol/wk7

Operon Construction  |  Glycobase

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**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

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.

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.

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

All samples were spun down.

Incubated all tubes for digestion period of 1 hour.

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,