Knight:DNA Spots

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(New page: ==Overview== ==Materials== ==Procedure== ===Making Spots=== ===Using spots to transform E. coli===)
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==Materials==
==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==
==Procedure==
===Making Spots===
===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===
===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.''

Revision as of 15:00, 26 July 2007

Contents

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.
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