Knight:DNA Spots: Difference between revisions
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**1% Cresol Red | **1% Cresol Red | ||
**DNA (100 ng/μL) | **DNA (100 ng/μL) | ||
**100% cotton, acid free thesis paper | **[http://www.crane.com/prdSell.aspx?Name=BT886S_ThesisPapers Crane & Co. 100% cotton, acid free thesis paper] | ||
*For using spots: | *For using spots to transform: | ||
**Harris Uni-Core Punch, 2mm and Olfa Cutting Mat | **Harris Uni-Core Punch, 2mm and Olfa Cutting Mat | ||
**TE | **[[TE buffer|TE]] | ||
**Competent Cells | **Competent Cells | ||
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===Making Spots=== | ===Making Spots=== | ||
*Mix 1 part 1% Cresol Red with 4 parts | [[Image:SpotGrid.jpg|thumb|left|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'' | **''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'' | ||
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===Using spots to transform E. coli=== | ===Using spots to transform E. coli=== | ||
[[Image:CuttingMat&Punch.jpg|thumb|left|Olfa cutting mat and Uni-Core Punch]] | |||
[[Image:Spot.jpg|thumb|left|Punched Spot]] | |||
*Cut out spot from surrounding paper using the Uni-Core Punch on the Olfa cutting mat | *Cut out spot from surrounding paper using the Uni-Core Punch on the Olfa cutting mat | ||
*Soak spot in 20 μL | *Soak spot in 20 μL TE for 15 minutes. | ||
*Thaw competent cells on ice while spots soak | *Thaw competent cells on ice while spots soak | ||
**''I used | **''I used [[TOP10 chemically competent cells]]'' | ||
*Add 5 μL of the TE the spot soaked in to 50 μL competent cells | *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.'' | **''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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**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. | **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 | *Incubate cells on ice for 2 minutes | ||
*Add 200 μL SOC | *Add 200 μL [[SOC]] | ||
*Incubate at 37 °C for 2 hours | *Incubate at 37 °C for 2 hours | ||
*Spread cells on previously made LB plates with proper antibiotic | *Spread cells on previously made LB plates with proper antibiotic | ||
*Grow overnight at 37 °C | *Grow overnight at 37 °C | ||
[[Category:Protocol]] [[Category:Escherichia coli]] | |||
Latest revision as of 06:29, 26 September 2007
Materials
- For making Spots:
- 1% Cresol Red
- DNA (100 ng/μL)
- 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
Procedure
Making Spots
- 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
- 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
