Imperial College/Courses/Spring2008/Synthetic Biology/Cellular And Molecular Biology Practical/Day 3

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Spring 2008 - Introduction to Synthetic Biology

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Wet Lab: EcoRV Restriction Endonuclease over expression and digestion of DNA




Schedule


From the stock solutions provided, make up the following buffers.

10 ml Buffer A

  • 50 mM Tris-HCl pH 7.5
  • 1 mM EDTA
  • 100 mM NaCl

1 ml 10x reaction buffer

  • 500 mM Tris pH 7.5
  • 1 M NaCl
  • 100 mM MgCl2
  1. Select the culture that had the greatest cell density at induction. Resuspend the cell pellet in the centrifuge tube in 1 ml of buffer A.
  2. Transfer to a 1.5 ml microcentrifuge tube (eppendorf) and add lysozyme to 1 mg/ml and leave on the bench for 15 min.
  3. Freeze the tube in the dry ice for 5 mins and then thaw in the 25°C waterbath for 5 mins. Repeat three times.
  4. Spin down the eppendorf in the microcentrifuge at full speed for 10 mins. (make sure the rotor is balanced)
  5. Prepare 4 x 1.5 ml tubes each with 10 l of 10x reaction buffer; 79 l H2O; 1 l BSA . Label 1-4.
  6. Take 10 l of the supernatant from what you have just spun down and add to tube 1.
  7. Mix thoroughly with a Gilson set at 100 l.
  8. Take 10 l from tube 1, add to tube 2 and mix. Repeat from tubes 2 to 3 and 3 to 4. (Question 3:: What fold dilution of your original cell extract do you have in your tubes 1-4?)


Setup a restriction digest before lunch.

  1. Into 4 x 1.5 ml tubes add 2l 10x reaction buffer; 15 l water; 0.5 l BSA; 1 g  DNA.

Note that when setting up enzyme reactons the order of addition of components is important. Enzymes must always be added to an appropriately buffered solution, never add enzyme to pure water or 10x buffer as this may well denature the protein and inactivate its enzymatic function.

  1. To each tube add 1 l of each diluted cell extract, mix and label.
  2. Place the tubes in a polystyrene float in the 37°C waterbath over lunch.


Run an agarose gel to analyse restriction fragments.

  1. 0.8% agarose gels are prepared by dissolving 0.2 g of agarose in 25 ml of buffer (TAE or TBE) by boiling. When it is cooling prepare the gel cast by applying tape to each end. When sufficiently cool (hand hot), SYBR green dye is added and the agarose is poured into a gel cast and a comb placed at one end.
  2. Add 4l of loading dye to each tube.
  3. When the gel is set remove the tape and place in an electrophoresis tank with a second gel from someone else. Make sure the gels are fully immersed in buffer.
  4. Load each reaction into the well of an agarose gel using a P20. (You will need to work with others to ensure that each gel is full).
  5. Run the gel at 50V for 20-30 mins or until the purple marker dye approaches the end of the gel.
  6. Visualise the restriction digest and take a picture.

Question 4: At what fold serial dilution do you see EcoRV activity ?

Question 5: 1 unit of enzyme activity is defined as being sufficient to digest one g of DNA in 1 h at 37°C. Approximately how many enzyme units per ml were in the original cell extract?

Question 6: 4000 units of EcoRV costs £41. How rich are you in EcoRV terms?

  • Write up your practical in a 2 page report detailing your results and answering the above questions. Reports should be as concise and factual as possible.
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