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Team:Newcastle University/Protocols
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Newcastle University
GOLD MEDAL WINNER 2008
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Isolating Plasmid from Cells (Miniprep)
Restricting Plasmids (Double Restriction)
Making Frozen Glycerol Cell Stocks
- Pellet overnight culture by centrifuging 13,000g for 10 minutes.
- Pipette out supernatent and discard.
- Add 250μl resuspension buffer R3 and mix by pipetting up and down.
- Transfer to capped tubes.
- Add 250μl lysis buffer L7 and mix by inverting tube 5 times.
- Incubate for 5 minutes at room temperature.
- Add 350μl precipitation buffer. Invert until mixture is homogenous.
- Centrifuge 13,000g for 10 minutes. Place spin column into recovery tube.
- Load supernatent into spin column and discard capped tube.
- Centrifuge 13,000g for 1 minute. Discard supernatent.
- Add 700μl wash buffer W9 (with ethanol).
- Centrifuge 13,000g for 1 minute. Discard superantent.
- Centrifuge 13,000g for 1 minute to remove all liquid. Discard any remaining supernatent and recovery tube.
- Place spin column in new recovery tube.
- Add 100μl TE buffer or MilliQ H2O.
- Incubate for 1 minute at room temperature.
- Centrifuge 13,000g for 2 minutes. The supernatent in the recovery tube should contain isolated plasmid.
- Discard spin column and store plasmid at -20°C.
Restricting Plasmids (Double Restriction)
We have conducted restrictions in varying concentrations and total volumes; however they all follow the same basic procedure. Below are two of the different restrictions we carried out.
- 46μl MillQ H2O
- 10μl 10 x buffer
- 40μl plasmid sample
- 2μl enzyme 1
- 2μl enzyme 2
Total volume = 100μl
Concentrated
- 8μl MilliQ H2O
- 5μl 10 X buffer
- 10μl plasmid sample
- 1μl enzyme 1
- 1μl enzyme 2
Total volume = 30μl
- Incubate solutions for 90 minutes in a 37°C water bath.
- If DNA is to be run on a gel and a band cut out, this can be done without purifying the DNA. For all other downstream applications or for storage, solution must be purified to remove the enzymes.
Ligation
To ligate our 2.2kb ncl08 fragment from pUC57-ncl08 into our 2 vector plasmids (pJWV021 and pGFP-rrnB), we followed the protocol below. Prior to ligation we did not isolate the 2.2kb fragment from the other resulting digest product (a 2.7kb fragment) because we will select for cells with the correct insert once we have transformed and plated the cells.
- 2.5μl of 10 x ligase buffer
- 6μl vector plasmid cut with relevant enzyme(s)
- 15μl of digested plasmid which contains the fragment to be inserted, digested with the same enzyme(s)as the vector plasmid
- 1.5μl of T4 ligase
= 25μl total volume
Control:
- 2.5μl of 10 x ligase buffer
- 6μl of vector plasmid cut with relevant enzyme(s)
- 15μl of MilliQ H2O
- 1.5μl of T4 ligase
= 25μl total volume
Transformation after ligation
- Add 2μl plasmid to 50μl of competent cells.
- Incubate for 30 minutes on ice.
- Heat shock for 2 minutes at 42°C.
- Add 250μl LB.
- Incubate for 120 minutes at 37°C whilst shaking or rotating.
Making Frozen Glycerol Cell Stocks
- Pippette 2μl overnight culture into an Eppendorf tube.
- Add 0.5ml glycerol and vortex briefly to mix.
- Prepare a freeze box by decanting a small amount of liquid nitrogen into the box. (N.B: wear protective gloves and face mask.)
- Place the tube in the box and keep at -80°C.
