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John Sy

Preparation for digestion of bacteria to isolate DNA for gel electrophoresis. We have identified 4 colonies on our plate containing our part I13273 which are potential cells with our desired DNA. However, only colonies 3 and 4 produced any bacteria. Media containing colonies 1 and 2 were still clear after leaving overnight.

Followed standard procedure for digestion (miniprep)

Formula for Restriction Enzyme addition after miniprep

  • 1 uL EcoRI
  • 1 uL PstI
  • 2 uL Buffer 0 (orange label)
  • 6 uL ultrapure water
  • 10 uL DNA sample

Heat in waterbath at 37C for approximately 30 minutes

Formula to make gel for electrophoresis

  • 0.2 g agarose in 20 mL 1x TAE
  • Place in microwave for about 1 minute to dissolve
  • Allow to cool to about 60C (warm enough to hold by hand)
  • add 1 uL ethidium bromide
  • pour into plates to make gels

Formula to prepare digested DNA for electrophroesis

  • Add 4 uL loading buffer containing RNAase
  • Transfer all 24 uL into one well
  • Run electrophoresis at about 80V, 0.5A

Results from digest

We seem to have obtained two different bands from the same restriction enzyme and DNA from two different wells. This is not what we expected. Looking at webcutter, we discover that our DNA part I13273 is not cut by either of the restriction enzymes. Dr. Mann suggests that the different banding may be due to supercoiling of the DNA.

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