Knight:DNA Spots

Materials

 * For making Spots:
 * 1% Cresol Red
 * DNA (100 ng/&mu;L)
 * 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

Making Spots

 * Mix 1 part 1% Cresol Red with 4 parts DNA that is at least 100 ng/&mu;L
 * The exact amounts depend on how many 2 &mu;L 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 &mu;L 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 &deg;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 &mu;L TE for 15 minutes.
 * Thaw competent cells on ice while spots soak
 * I used TOP10 chemically competent cells
 * Add 5 &mu;L of the TE the spot soaked in to 50 &mu;L 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 &deg;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 &mu;L SOC
 * Incubate at 37 &deg;C for 2 hours
 * Spread cells on previously made LB plates with proper antibiotic
 * Grow overnight at 37 &deg;C