Knight:DNA Spots: Difference between revisions
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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 13:00, 26 July 2007
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.
