Difference between revisions of "User:KCliff/Evaluating Cross Contamination Using The Pin Tool"
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1.  Add 1.0 ml of 25% Cresol Red solution to the wells of every other column on the deep well plate.
 
1.  Add 1.0 ml of 25% Cresol Red solution to the wells of every other column on the deep well plate.
  
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2.  [[Transfer]] the 25% Cresol Red solution to the thesis paper grid using the Pin Tool Replicator.
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2.  [[User:KCliff/Pin_Tool/Transfer|Transfer]] the 25% Cresol Red solution to the thesis paper grid using the Pin Tool Replicator.
  
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3.  [[Clean]] the pin tool replicator.
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3.  [[User:KCliff/Pin_Tool/Clean|Clean]] the pin tool replicator.
  
 
4.  Observe spotting on thesis paper grid to determine cross contamination among grid spaces.
 
4.  Observe spotting on thesis paper grid to determine cross contamination among grid spaces.
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*96 deep well plate (2)
 
*96 deep well plate (2)
 
*96 Multi-Blot (Pin Tool) Replicator
 
*96 Multi-Blot (Pin Tool) Replicator
 +
*Library Copier (2)
 
*LB with Ampicillin broth
 
*LB with Ampicillin broth
 
*Red Florescence Protein (RFP) cells
 
*Red Florescence Protein (RFP) cells
−
*Library Copier (2)
 
 
*Lint-free blotting paper  
 
*Lint-free blotting paper  
 
*VP 110 Pin Cleaning Solution
 
*VP 110 Pin Cleaning Solution
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2.  To every other column of wells add 10. ul of suspended RFP cells, leaving the alternate columns with only LB broth.   
 
2.  To every other column of wells add 10. ul of suspended RFP cells, leaving the alternate columns with only LB broth.   
  
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3.  Using the Pin Tool Replicator, [[Transfer]] the culture from the source plate to a transfer 96 deep well plate of LB broth.
+
3.  Using the Pin Tool Replicator, [[User:KCliff/Pin_Tool/Transfer|Transfer]] the culture from the source plate to a transfer 96 deep well plate of LB broth.
  
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4.  [[Clean]] the Pin Tool Replicator.
+
4.  [[User:KCliff/Pin Tool/Clean|Clean]] the Pin Tool Replicator.
  
 
5.  Plate four random samples from the columns containing only LB broth and incubate overnight.
 
5.  Plate four random samples from the columns containing only LB broth and incubate overnight.
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*96 deep well plate (4)
 
*96 deep well plate (4)
 
*96 Multi-Blot (Pin Tool) Replicator
 
*96 Multi-Blot (Pin Tool) Replicator
 +
*Library Copier (2)
 
*LB with Ampicillin broth
 
*LB with Ampicillin broth
 
*Red Florescence Protein (RFP) cells
 
*Red Florescence Protein (RFP) cells
−
*Library Copier (2)
 
 
*Lint-free blotting paper  
 
*Lint-free blotting paper  
 
*VP 110 Pin Cleaning Solution
 
*VP 110 Pin Cleaning Solution
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3.  Remain with two plates that will act as first and second transfer plates.     
 
3.  Remain with two plates that will act as first and second transfer plates.     
  
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4.  Using the Pin Tool Replicator, [[Transfer]] the culture from the first source plate to the first transfer plate.
+
4.  Using the Pin Tool Replicator, [[User:KCliff/Pin_Tool/Transfer|Transfer]] the culture from the first source plate to the first transfer plate.
  
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5.  [[Clean]] the Pin Tool Replicator.
+
5.  [[User:KCliff/Pin Tool/Clean|Clean]] the Pin Tool Replicator.
  
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6.  Then, use the Pin Tool Replicator to [[transfer]] from the second source plate to the second transfer plate.
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6.  Then, use the Pin Tool Replicator to [[User:KCliff/Pin_Tool/Transfer|Transfer]] from the second source plate to the second transfer plate.
  
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7.  [[Clean]] the Pint Tool Replicator.
+
7.  [[User:KCliff/Pin Tool/Clean|Clean]] the Pint Tool Replicator.
  
 
8.  Plate four random samples from the second transfer plate and incubate overnight.
 
8.  Plate four random samples from the second transfer plate and incubate overnight.
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*96 deep well plate (3)
 
*96 deep well plate (3)
 
*96 Multi-Blot (Pin Tool) Replicator
 
*96 Multi-Blot (Pin Tool) Replicator
 +
*Library Copier
 
*LB with Ampicillin broth
 
*LB with Ampicillin broth
 
*LB with Ampicillin/Kanamycin
 
*LB with Ampicillin/Kanamycin
 
*Red Fluorescence Protein (RFP) cells
 
*Red Fluorescence Protein (RFP) cells
−
*Library Copier (2)
 
 
*Lint-free Blotting Paper
 
*Lint-free Blotting Paper
 
*VP 110 Pin Cleaning Solution
 
*VP 110 Pin Cleaning Solution
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2.  Add 10. ul of suspended RFP cells into each well of the source plate.
 
2.  Add 10. ul of suspended RFP cells into each well of the source plate.
  
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3.
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3.  Prepare the other two 96 deep well source plates, with LB broth with Ampicillin/Kanamycin in the first and LB broth with Ampicillin only in the second.
 +
 
 +
4.  Using the Pin Tool Replicator, [[User:KCliff/Pin_Tool/Transfer|transfer]] the culture from the source plate to the first deep well plate containing LB broth with Ampicillin/Kanamycin.
 +
 
 +
5.  [[User:KCliff/Pin Tool/Clean|Clean]] the Pin Tool Replicator.
 +
 
 +
6.  Then, use the Pin Tool Replicator to inoculate the second source plate, containing LB broth with Ampicillin only, with source plate culture.
 +
 
 +
7.  Incubate and plate samples from the second antibiotic testing plate.
 +
 
 +
8.  Observe culture growth.

Latest revision as of 19:54, 28 June 2017

In preparation for the 2008 iGEM competition, selected DNA 'bioparts' found in the Registry are transferred onto sheets of thesis paper using the pin tool replicator in order to be mailed to teams at participating colleges and universities. Since this is the first year that iGEM will be using the thesis paper in place of welled plates to distribute the 'bioparts', the pin tool replicator method of transfer and cleaning was evaluated for possible DNA and culture cross contamination.


Cross Contamination Among DNA Samples from the Same Source Plate

Materials:

  • 96 deep well plate
  • 25% Cresol Red solution
  • 96 Multi-Blot (Pin Tool) Replicator
  • source plate Library Copier
  • Thesis Paper with iGEM 96-block grid print
  • Lint-free blotting paper
  • VP 110 Pin Cleaning Solution
  • 99% Isopropynol


1. Add 1.0 ml of 25% Cresol Red solution to the wells of every other column on the deep well plate.

2. Transfer the 25% Cresol Red solution to the thesis paper grid using the Pin Tool Replicator.

3. Clean the pin tool replicator.

4. Observe spotting on thesis paper grid to determine cross contamination among grid spaces.


Culture Cross Contamination Among Samples from the Same Source Plate

Materials:

  • 96 deep well plate (2)
  • 96 Multi-Blot (Pin Tool) Replicator
  • Library Copier (2)
  • LB with Ampicillin broth
  • Red Florescence Protein (RFP) cells
  • Lint-free blotting paper
  • VP 110 Pin Cleaning Solution
  • 99% Isopropynol
  • LB Agar plates with Ampicillin


1. Add 500. ul of LB broth to each well in a 96-deep well source plate.

2. To every other column of wells add 10. ul of suspended RFP cells, leaving the alternate columns with only LB broth.

3. Using the Pin Tool Replicator, Transfer the culture from the source plate to a transfer 96 deep well plate of LB broth.

4. Clean the Pin Tool Replicator.

5. Plate four random samples from the columns containing only LB broth and incubate overnight.

6. Observe for growth of RFP cells in the samples where only LB broth should have been present.


Culture Cross Contamination Among Different Source Plates

Materials:

  • 96 deep well plate (4)
  • 96 Multi-Blot (Pin Tool) Replicator
  • Library Copier (2)
  • LB with Ampicillin broth
  • Red Florescence Protein (RFP) cells
  • Lint-free blotting paper
  • VP 110 Pin Cleaning Solution
  • 99% Isopropynol
  • LB Agar plates with Ampicillin


1. Add 500. ul of LB broth to each well of four 96 deep well plates.

2. To the first source plate, add 10. ul of suspended RFP cells to each well, and leave the second source plate and with only LB broth.

3. Remain with two plates that will act as first and second transfer plates.

4. Using the Pin Tool Replicator, Transfer the culture from the first source plate to the first transfer plate.

5. Clean the Pin Tool Replicator.

6. Then, use the Pin Tool Replicator to Transfer from the second source plate to the second transfer plate.

7. Clean the Pint Tool Replicator.

8. Plate four random samples from the second transfer plate and incubate overnight.

9. Observe for growth of RFP cells in samples where only LB broth should be present.


Antibiotic Cross Contamination Among Different Antibiotic Testing Plates

Materials:

  • 96 deep well plate (3)
  • 96 Multi-Blot (Pin Tool) Replicator
  • Library Copier
  • LB with Ampicillin broth
  • LB with Ampicillin/Kanamycin
  • Red Fluorescence Protein (RFP) cells
  • Lint-free Blotting Paper
  • VP 110 Pin Cleaning Solution
  • 99% Isopropynol
  • LB Agar plates with Ampicillin


1. Add 500. ul of LB broth to each well of one 96 deep well plate.

2. Add 10. ul of suspended RFP cells into each well of the source plate.

3. Prepare the other two 96 deep well source plates, with LB broth with Ampicillin/Kanamycin in the first and LB broth with Ampicillin only in the second.

4. Using the Pin Tool Replicator, transfer the culture from the source plate to the first deep well plate containing LB broth with Ampicillin/Kanamycin.

5. Clean the Pin Tool Replicator.

6. Then, use the Pin Tool Replicator to inoculate the second source plate, containing LB broth with Ampicillin only, with source plate culture.

7. Incubate and plate samples from the second antibiotic testing plate.

8. Observe culture growth.