DNA Spots-PDF

Materials

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

Spotted Grid

Mix 1 part 1% Cresol Red with 4 parts DNA that is at least 100 ng/μL
- 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.

Olfa cutting mat and Uni-Core Punch
Punched Spot

Cut out spot from surrounding paper using the Uni-Core Punch on the Olfa cutting mat
Soak spot in 20 μL 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 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