Knight:DNA Spots

Materials

• For making Spots:
  • 1% Cresol Red
  • DNA at a concentration between 10 and 100 ng/μL
  • 100% cotton, acid free thesis paper
• For using spots:
  • Micro Punch or some other method of removing excess paper surrounding dried DNA
  • 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
• 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
  • I used a Micro Punch for this - it cuts out circles with a 2 mm diameter
• Soak spot in 20 μL 10:1 TE for 15 minutes.
• Add 5 μL of the TE the spot soaked in to 50 μL competent cells to be transformed
  • 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.