Team:Exeter/lab book/novpol/wk9

Operon Construction  |  Glycobase

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**Monday 03/09/12**

9.00am

Transformations using Gene_Terminator.

* Tetr_RBS + Cyc_Term

* Tetr_RBS + HAS_Term

* ompA + sacB_Term

11.20am

All in the incubator for digestion period.

12.40pm

Heat denatured enzymes at 80°C for 20 minutes.

1.00pm

Ligation process

Plasmid + Upstream + Downstream

Plasmid + Tetr_RBS + Cyc_Term

Plasmid + Tetr_RBS + HAS_Term

Plasmid + OmpA + SacB_Term

2.50pm

Transformations

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

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 prep. However 40μl of clean water was added, instead of the previous 50μl, to boost the concentration of DNA in each sample.

1.30pm

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

Into incubator for overnight digestion.

**Friday 07/09/12**

11.00am

Gel electrophoresis run.

Sequences confirmed for:

Cyclodextrin_Terminator

HAS_Terminator

SacB_Terminator

Single genes confirmed for:

Cyclodextrin

HAS