Knight:DNA Spots

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Current revision (08:29, 26 September 2007) (view source)
(Making Spots)
 
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===Making Spots===
===Making Spots===
[[Image:SpotGrid.jpg|thumb|left|Spotted Grid]]
[[Image:SpotGrid.jpg|thumb|left|Spotted Grid]]
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*Mix 1 part 1% Cresol Red with 4 parts DNA that is at least 100 μL/ng
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*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''
**''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''
**''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''

Current revision

Contents

Materials

Procedure

Making Spots

Spotted Grid
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.

Using spots to transform E. coli

Olfa cutting mat and Uni-Core Punch
Olfa cutting mat and Uni-Core Punch
Punched Spot
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
  • 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
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