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	=Standard Protocols=		=Standard Protocols=
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	This page provides a database of all the standard protocols used throughout our project. Some procedures are directly listed, whilst those provided by commercial kits are simply linked to.		This page provides a database of all the standard protocols used throughout our project. Some procedures are directly listed, whilst those provided by commercial kits are simply linked to.

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Revision as of 18:00, 27 October 2010

Standard Protocols

This page provides a database of all the standard protocols used throughout our project. Some procedures are directly listed, whilst those provided by commercial kits are simply linked to.

Making Competent Cells

We used this procedure to make both competent E. coli MG1655 and XL1-blue. Where we were used particularly small amounts of DNA we used commercial NovaBlue cells.

1. Inoculate 50ml Lennox broth in a 150ml flask with 1ml of an overnight culture of E. coli.
2. Incubate at 37°C until mid-logarithmic phase (A600 ~0.35, approx 2h (we checked after 1h30))
3. Transfer culture to a sterile, chilled, centrifuge tube. Incubate on ice for 10min
4. Harvest cells by 5min. centrifugation at 3600rpm at 4°C. Discard supernatant.
5. Resuspend cell pellet in 20ml of ice cold 0.1M CaCl2 Incubate on ice for 20min
6. Harvest cells by 5min. centrifugation at 3600rpm at 4°C. Discard supernatant.
7. Either: Resuspend pellet in 5ml ice cold CaCl2. Incubate on ice for at least 30mins prior to use (competence will continue to increase for up to 24h if cells stored on ice. Or:
8. Resuspend pellet in 3ml ice-cold 0.1M CaCl2 plus 2ml ice-cold 50%glycerol. Incubate on ice for 30min then store in aliquots at -80°C.

Transforming Competent Cells

This procedure is for the transformation of cells rendered competent by the procedure above. When using NovaBlues a different, quicker method can be followed.

1. Add the DNA to be transformed to an empty, chilled microfuge tube
2. Add 100μl of icecold competent E. coli
3. Incubate on ice for 30mins (or longer)
4. Heat shock by placing at 42°C got 2 mins
5. Add 0.5ml of Lennox broth and incubate at 37°C for 30-90mins
6. Pellet the cells by spinning briefly in a minifuge. Discard the supernatant
7. Resuspend each pellet in 100μl of Lennox broth and plate out onto an agar plate containing appropriate antibiotics for plasmid selection. Incubate the plate at 37°C overnight.

Transforming NovaBlues

This procedure should only be used when transforming commercial competent cells.

1. Thaw required number of cell tubes on ice, mix gently to ensure suspension
2. Add 5mL DNA to 50μL cells, stir gently to mix
3. Place tubes on ice for 5 minutes
4. Heat shock for 30s at 42°C
5. Place tubes on ice for 2 minutes
6. Add 250μL of SOC medium
7. Plate out 100μL of cell suspension, incubate overnight at 37°C

Minipreps, Midipreps and Gel Extractions

Gel extractions, Mini and midipreps were performed using Qiagen kits, following the procedures provided by them:

[http://www.qiagen.com/literature/render.aspx?id=370 Miniprep]

[http://www.qiagen.com/literature/render.aspx?id=369 Midiprep]

[http://www.qiagen.com/literature/render.aspx?id=103715 Gel Extraction]