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

Making a gel

To make a 1.5L buffer:

1. Measure 30mL of 50x TAE buffer
2. Transfer to a 2L measuring cylinder
3. Fill up to 1.5L with distilled water

To make a 1% agarose gel:

1. Weigh out 1g of agarose powder
2. Transfer to a microwave bottle
3. Pour 100mL of your buffer into the bottle
4. Microwave until clear
5. Cool down to 55⁰C before pouring the gel

Gel electrophoresis

To run the gel:

1. Place the gel in the tank and cover it with the buffer
2. Load 5μl of DNA ladder into the 2nd well (leave the first one empty)
3. Add 5μl of loading dye (30% glycerol, 1% bromophenol blue, 0.5% sodium dodecyl sulphate, diluted in TE buffer) into each restriction digest and pipette up and down a few times
4. Load 25μl of each restriction digest into a separate well
5. Run gel at 1.5 Amp, 100V for approximately 1 hour

To stain the gel: Ethidium bromide: stain in (concentration) for 40 minutes, destain for 40 minutes, image under UV light

Azure A (for gel extraction): stain in 0.04%/20% ethanol for 15 minutes, destain for 15 minutes (multiple rounds of destaining may be required), image under visible light

Gel extraction using Qiagen kit=

1. Weigh an empty eppendorf and record the weight
2. Cut out a the band from the gel and place it in the eppendorf
3. Weigh the eppendorf again and calculate the weight difference
4. Add 3 volumes of Buffer QG to 1 volume of gel
5. Incubate at 50⁰C for 10 minutes until the agarose has dissolved. Vortex every 2-3 minutes
6. Add 1 volume of isopropanol and mix
7. Transfer the sample from the eppendorf to a QIAprep spin column in a 2mL collection tube
8. Centrifuge for 1 minute at 13,000rpm and discard flowthrough
9. Add 500μl of Buffer QG to column
10. Centrifuge for 1 minute at 13,000rpm and discard flowthrough
11. Add 750μl of Buffer PE and let it stand for 2-5 minutes
12. Centrifuge for 1 minute at 13,000rpm and discard flowthrough. Repeat this step
13. Transfer the column to a new eppendorf and discard the collection tube
14. Add 30μl of Buffer EB to the sample and let it stand for 1 minute
15. Centrifuge for 1 minute at 13,000rpm and keep discard the column

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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