Difference between revisions of "User:KCliff/Evaluating Cross Contamination Using The Pin Tool"
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'''Culture Cross Contamination Among Different Source Plates'''
 
'''Culture Cross Contamination Among Different Source Plates'''
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Materials:
 
Materials:
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*VP 110 Pin Cleaning Solution
 
*VP 110 Pin Cleaning Solution
 
*99% Isopropynol
 
*99% Isopropynol
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1.  Add 500. ul of LB broth to each well of four 96-deep well plates.
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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.
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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.
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5.  [[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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7.  [[Clean]] the Pint Tool Replicator.
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8.  Plate four random samples from the second transfer plate and incubate overnight.
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9.  Observe for growth of RFP cells in samples where only LB broth should be present.

Revision as of 20:31, 15 February 2008

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 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
  • LB with Ampicillin broth
  • Red Florescence Protein (RFP) cells
  • Library Copier (2)
  • Lint-free blotting paper
  • VP 110 Pin Cleaning Solution
  • 99% Isopropynol


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
  • LB with Ampicillin broth
  • Red Florescence Protein (RFP) cells
  • Library Copier (2)
  • Lint-free blotting paper
  • VP 110 Pin Cleaning Solution
  • 99% Isopropynol


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.