Knight:DNA Spots
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Overview
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.