Knight:DNA Spots

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Revision as of 14:33, 24 August 2007 by Samantha L. Burke (Talk | contribs)
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Contents

Materials

  • For making Spots:
    • 1% Cresol Red
    • DNA (100 ng/μL)
    • 100% cotton, acid free thesis paper
  • For using spots:
    • Harris Uni-Core Punch, 2mm and Olfa Cutting Mat
    • TE
    • Competent Cells

Procedure

Making Spots

  • Mix 1 part 1% Cresol Red with 4 parts 10-100 ng/μL DNA
    • The exact amounts depend on how many 2 μL spots you plan to make
    • Using the excess of Cresol Red is helpful for when you transform, since you can visibly tell which cells have had DNA added and which have not
  • Make 2 μL spots on 100% cotton, acid free thesis paper
    • Place a second sheet of paper under the one to be spotted to keep it free from contamination
  • Leave spots to dry at room temperature. This takes between 45 minutes and an hour
  • Once dry, spots can be used right away or stored at -20 °C. To store - place the spotted paper between two others to protect it.

Using spots to transform E. coli

  • Cut out spot from surrounding paper using the Uni-Core Punch on the Olfa cutting mat
  • Soak spot in 20 μL 10:1 TE for 15 minutes.
  • Thaw competent cells on ice while spots soak
    • I used Top10 chemically competent cells
  • Add 5 μL of the TE the spot soaked in to 50 μL competent cells
    • Spots could also be directly added to cells. Soaking in TE is better though, since there is DNA left over if the transformation does not work for some reason.
  • Incubate cells on ice for 30 minutes
  • Heat shock cells at 43 °C
    • If cells are in individual tubes, heat shock for 45 seconds. If cells are in a 96-well plate extend the heat shock to 1 minute.
  • Incubate cells on ice for 2 minutes
  • Add 200 μL SOC
  • Incubate at 37 °C for 2 hours
  • Spread cells on previously made LB plates with proper antibiotic
  • Grow overnight at 37 °C
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