Difference between revisions of "Team:Glasgow/Protocols"

Preparation of CaCl2 competent cells

1. Dilute 500μl of overnight liquid culture into 20ml of broth with any necessary antibiotics to select for any plasmids already transformed into the cells.
2. Incubate at 37⁰C, shaking at 225rpm for 105 minutes.
3. Spin down for 2 minutes at 7000G at 4⁰C.
4. Discard supernatant, resuspend pellet in 10ml of 50 mM CaCl2, keep on ice.
5. Repeat centrifugation for 2 minutes at 7000G at 4⁰C.
6. Discard supernatant and the resuspend pellet in 1ml of 50 mM CaCl2, keep on ice.
7. CaCl2 competent cells can be kept on ice in the fridge overnight.

Transformation of CaCl2 competent cells

1. Add 1μl of plasmid DNA to 100μl of competent cells.
2. Heat shock at 42oC for 45 seconds.
3. Add 200μl of L-broth of the sample.
4. Keep on ice for 2 minutes.
5. Incubate the cells at 37oC. The time varies depending on which antibiotic resistance the plasmid holds.

Figure 1: Incubation time for transformed cells containing different antibiotic resistances

Restriction digests

A 20μl reaction typically contained:

1. 2μl buffer
2. 4μl Miniprep (or 8μl G-Block) dependant on concentration of Miniprep
3. Make up to 20μl with ddH20
4. Vortex briefly.
5. Incubate at 37oC for at least 60 minutes.
6. Heat inactivate restriction digests where appropriate.

Miniprep

1. Pipette 1mL of bacterial overnight culture into a microcentrifuge tube
2. Centrifuge at 13,000 rpm for 1 min
3. Discard the supernatant
4. Repeat steps 1-3 two more times
5. Add 250μl of P1 buffer and pipette up and down to resuspend the pellet
6. Add 250μl of P2 buffer and invert Note: don’t allow this lysis reaction to proceed for more than 5 minutes
7. Add 350μl of N3 buffer to neutralise the reaction and invert
8. Centrifuge at 13,000 rpm for 10 minutes
9. Transfer 800μl of supernatant into a column
10. Centrifuge for 1 minute and discard flow-through
11. Add 500μl of PB buffer and centrifuge for 1 minute
12. Discard flow-through
13. Add 750μl of PE buffer and centrifuge for 1 minute
14. Discard flow-through
15. Centrifuge again for 1 minute to get rid of any residual buffer
16. Transfer the column to a microcentrifuge tube
17. Add 50μl of EB buffer and let it stand for 1 minute
18. Centrifuge for 1 minute

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Materials and Methods

Condition set up

Sample preparation

Table 1: Optical density analysis of S. thermophilus growth

Results and Discussion

Outlook

References