Team:St Andrews/Lab-book

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                   GATC sequencing
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                   Transformation
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                Sequencing samples should be in water solution rather than EB (can do another clean up ending up with     water instead of EB)
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<p>1.      Let frozen cell thaw from (-80°C) and warm only with finger tips when using </p>
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collect at 12 noon
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<p>2.      Mix 10uL of cells with 1uL of vectors, and leave on ice for 30 min.</p>
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use Chris’ usename to log in via GATC website, use a label online and stick the corresponding one onto the side of sample tubes
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<p>3.      Put 1 ml of LB liquid medium into Eppendorf tube, warm up briefly in 37°C</p>
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choose from the primers the company provides according to needs
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<p>4.      Put the cell and vector mixture on 42°C water bath for 45s to heat shock the cells, allowing membrane holes to close</p>
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place the order and then leave the box with the sample tube at old BMS building reception, there is GATC collection box.
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<p>5.      Pipette 50uL of warm LB liquid medium into the tube with mixture</p>
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<p>6.      Shake at 37°C for 30 min; recombinant <i>E.coli</i> will start to grow</p>
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<p>7.      Spread all contents of the shaken tube onto agar plate and incubate at 37°C overnight<p>
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8.      Pipette using 10ml pipette and pipette gun to transfer 10-15ml (leave enough air for bacterial growth) of liquid LB medium in four 50ml Falcon tubes; one labelled tube per colony
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9.      Harvest recombinant bacteria from agar plate by scratching gently with the scraper. The plate should be facing downwards to minimise contamination.
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10.  Put the scraper tip into the Falcon tubes to transfer the bacteria into medium
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11. Add into the Falcon tubes 1uL of ampicillin (1uL for 10uL)
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12.  Incubate the bacteria on shaker incubator at 37°C overnight but not excess 20hrs (otherwise the plasmids will be expelled from the cells)
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Revision as of 09:03, 9 July 2012

St Andrews iGEM '12

Lab Book

Protocols

Transformation

1. Let frozen cell thaw from (-80°C) and warm only with finger tips when using

2. Mix 10uL of cells with 1uL of vectors, and leave on ice for 30 min.

3. Put 1 ml of LB liquid medium into Eppendorf tube, warm up briefly in 37°C

4. Put the cell and vector mixture on 42°C water bath for 45s to heat shock the cells, allowing membrane holes to close

5. Pipette 50uL of warm LB liquid medium into the tube with mixture

6. Shake at 37°C for 30 min; recombinant E.coli will start to grow

7. Spread all contents of the shaken tube onto agar plate and incubate at 37°C overnight

8. Pipette using 10ml pipette and pipette gun to transfer 10-15ml (leave enough air for bacterial growth) of liquid LB medium in four 50ml Falcon tubes; one labelled tube per colony 9. Harvest recombinant bacteria from agar plate by scratching gently with the scraper. The plate should be facing downwards to minimise contamination. 10. Put the scraper tip into the Falcon tubes to transfer the bacteria into medium 11. Add into the Falcon tubes 1uL of ampicillin (1uL for 10uL) 12. Incubate the bacteria on shaker incubator at 37°C overnight but not excess 20hrs (otherwise the plasmids will be expelled from the cells)

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