Team:Newcastle University/Protocols

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Newcastle University

GOLD MEDAL WINNER 2008

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Agarose Gel Electrophoresis

Isolating Plasmid from Cells (Miniprep)

Restricting Plasmids (Double Restriction)

Ligation

Transformation

Making Frozen Glycerol Cell Stocks


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.