Knight:DNA Spots

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Current revision (08:29, 26 September 2007) (view source)
(Making Spots)
 
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==Overview==
 
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==Materials==
==Materials==
*For making Spots:
*For making Spots:
**1% Cresol Red
**1% Cresol Red
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**DNA at a concentration between 10 and 100 ng/μL
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**DNA (100 ng/μL)
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**100% cotton, acid free thesis paper
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**[http://www.crane.com/prdSell.aspx?Name=BT886S_ThesisPapers Crane & Co. 100% cotton, acid free thesis paper]
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*For using spots:
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*For using spots to transform:
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**Micro Punch or some other method of removing excess paper surrounding dried DNA
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**Harris Uni-Core Punch, 2mm and Olfa Cutting Mat
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**TE
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**[[TE buffer|TE]]
**Competent Cells
**Competent Cells
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===Making Spots===
===Making Spots===
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*Mix 1 part 1% Cresol Red with 4 parts 10-100 ng/μL DNA
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[[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 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''
*Make 2 μL spots on 100% cotton, acid free thesis paper
*Make 2 μL spots on 100% cotton, acid free thesis paper
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**''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''
*Leave spots to dry at room temperature. ''This takes between 45 minutes and an hour''
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*Once dry, spots can be used right away or stored at -20 °C
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*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===
===Using spots to transform E. coli===
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*Cut out spot from surrounding paper
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[[Image:CuttingMat&Punch.jpg|thumb|left|Olfa cutting mat and Uni-Core Punch]]
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**''I used a Micro Punch for this - it cuts out circles with a 2 mm diameter''
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[[Image:Spot.jpg|thumb|left|Punched Spot]]
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*Soak spot in 20 μL 10:1 TE for 15 minutes.  
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*Cut out spot from surrounding paper using the Uni-Core Punch on the Olfa cutting mat
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*Add 5 μL of the TE the spot soaked in to 50 μL competent cells to be transformed
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*Soak spot in 20 μL TE for 15 minutes.  
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*Thaw competent cells on ice while spots soak
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**''I used [[TOP10 chemically competent cells]]''
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*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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*Incubate cells on ice for 30 minutes
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*Heat shock cells at 43 °C
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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.
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*Incubate cells on ice for 2 minutes
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*Add 200 μL [[SOC]]
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*Incubate at 37 °C for 2 hours
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*Spread cells on previously made LB plates with proper antibiotic
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*Grow overnight at 37 °C
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[[Category:Protocol]] [[Category:Escherichia coli]]

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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