Knight:DNA Spots

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

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 30 seconds, if 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