Laboratory Work at the Kanbar Lab

• March 1, 2012 (Start time: 15:15)
• Procedure:
  1. Incubate mini culture of bacteria with transformed gene at 37C, 200rpm shaking overnight

• March 2, 2012 (Start time: 13:15)
• Sample name as follows:
  1. Sample 1: Bacteria, CdCl2, Na2S, IPTG, 3 hrs.
  2. Sample 2: No bacteria, CdCl2, Na2S, IPTG, 3 hrs.
  3. Sample 3: Bacteria, CdCl2, Na2S, IPTG, 2 hrs.
  4. Sample 4: Bacteria, CdCl2, Na2S, IPTG, 1 hr.
  5. Sample 5: Bacteria, CdCl2, IPTG, 3 hrs.
  6. Sample 6: Bacteria, Na2S, IPTG, 3 hrs.
  7. Sample 7: Bacteria, CdCl2, Na2S, IPTG, 3 hrs.
• Procedure:
  1. Warmed the pre-prepared IPTG solution
  2. Weighed .0109 g CdCl2
  3. Added 10 μL IPTG into samples 1-6
  4. Measured 120 μL of deionized H2O in preparation for CdCl2 solution
  5. Inserted 20 μL of 1 mM solution of CdCl2 to samples 1-5, 7 (13:28)
  6. Placed all samples into orbital rocker (belly-dancer) for 60 minutes.
  7. Weighed .0145 g Na2S⋅9H2O
  8. Measured 20 μL of deionized H2O in preparation for Na2S solution
  9. Added 20 μL of Na2S⋅9H2O to sample 4 (final concentration: 1mM) and returned sample to orbital rocker for 90 minute incubation (14:40)
     • Observed no significant color change.
  10. Added 20 μL of Na2S⋅9H2O to sample 3 (final concentration: 1mM) and returned sample to orbital rocker for 90 minute incubation (15:35)
      • Observed no significant color change.
  11. Removed sample 4 and centrifuged for 5 min. at 1000rpm (16:03)
      • Observed significant cell suspension.
      • Decided to increase the rotation to 2000rpm.
  12. Centrifuged sample 4 for additional 5 min. at 2000rpm (16:09)
      • Observed significant cell suspension.
      • Decided to increase the rotation to 3000rpm.
  13. Centrifuged sample 4 three times for additional 5 min. each at 3000rpm and washed sample after each centrifugation (16:17)
      • Observed some cell suspension.
      • Agreed that sample was at the centrifuge's limit.
      • Concluded optimal centrifusion should be conducted at 3000rpm for 5 min.
  14. Centrifuged sample 5 three times for 5 min. each at 3000rpm and washed sample after each centrifugation (16:35)
  15. Added 20 μL of Na2S⋅9H2O to samples 1,2,6,7 (final concentration: 1 mM) and returned samples to orbital rocker for 90 minute incubation (16:40)
      • Observed no significant color change.
  16. Stored sample 4 at -80C (16:42)
  17. Stored sample 5 at -80C (17:02)
  18. Centrifuged sample 3 three times for 5 min. each at 3000rpm and washed sample after each centrifugation (17:03)
  19. Stored sample 3 at -80C (17:27)
  20. Centrifuged samples 1,6,7 three times for 5 min. each at 3000rpm and washed sample after each centrifugation (18:02)
  21. Centrifuged sample 2 for 5 min. at 3000 rpm (18:02)
  22. Stored sample 2 at -80C (18:12)
  23. Stored samples 1,6,7 at -80C (18:33)
• March 2, 2012 (End time: 18:35)

• March 28, 2012 (Start time: 10:05)
• Procedure:
  1. Plated pET28+CDS7 with Kanamycin (4μL)
  2. Plated Red Luciferase+LRE with Chloramphenicol (4μL)
  3. Plated PS1C3 with Chloramphenicol (4μL)
  4. Left plates to incubate overnight at 37C
• Note:
  1. The pET28+CDS7 bacteria contain CDS7, the metal binding protein. The Red Luciferase+LRE and the PS1C3 bacteria contain the chloramphenicol resistant backbones. Eventually, these vectors will be extracted from their respective plasmids and will be combined to form plasmids that contain CDS7 and chloramphenicol resistance.
• March 28, 2012 (End time: 10:35)

• March 29, 2012 (Start time: 13:00)
• Procedure:
  1. Prepared 9 test tubes with LB broth
  2. Designated 3 for pET28+CDS7 with Kanamycin (labeled test tube set A)
  3. Designated 3 for PS1C3 with Chloramphenicol (labeled test tube set B)
  4. Designated 3 for Red Luciferase+LRE with Chloramphenicol (labeled test tube set C)
  5. Added 5 μL of respective antibiotic in each test tubes
  6. Added 1 colony of pET28+CDS7 containing bacteria in each test tube of set A
  7. Added 1 colony of PS1C3 containing bacteria in each test tube of set B
  8. Added 1 colony of Red Luciferase+LRE containing bacteria in each test tube of set C
     • Note: For test tube set A, since only 3 colonies formed during the overnight incubation, the colonies were streaked and extracted.
  9. Incubated the test tubes in reciprocating water bath at 120 rpm overnight
• March 29, 2012 (End time: 13:25)

• April 3, 2012 (Start time: 14:05)
• Note: Prior to experiment for the day, observed a significant loss in live bacteria in samples.
• Procedure:
  1. Prepared 9 test tubes with LB broth
  2. Replaced labeled caps from test tubes of Day 4 to caps of new test tubes
  3. Added 5 μL of respective antibiotic in each test tubes
  4. Added 100 μL of surviving pET28+CDS7 containing bacteria from day 4 test tube in each new test tube of set A
  5. Added 100 μL of surviving PS1C3 containing bacteria from day 4 test tube in each new test tube of set B
  6. Added 100 μL of surviving Red Luciferase+LRE containing bacteria from day 4 test tube in each new test tube of set C
  7. Incubated the test tubes in reciprocating water bath at 120 rpm overnight
• April 3, 2012 (End time: 14:45)

• April 4, 2012 (Start time: 10:05)
• Procedure:
  1. Obtained 4 microcentrifuge tubes.
  2. Labeled two tubes A and two tubes B for respective samples
  3. Filled each microcentrifuge tube with 1.5 mL of respective bacterial culture
  4. Centrifuged all 4 samples at 13,000 rpm for 3 minutes
  5. Decanted the supernatant
  6. Resuspended the bacterial pellets in 0.25 mL of Buffer P1
  7. Added 0.25 mL of Buffer P2 after resuspension
  8. Mixed the solution by inverting tube until solution was homogeneous blue
  9. Incubated samples for no more than 3 minutes
  10. Added 0.35 mL of Buffer N3
  11. Mixed the samples immediately until solution becomes colorless
  12. Centrifuged samples for 10 minutes at 11,000 rpm
  13. Transferred the supernatant to spin column
  14. Centrifuged for additional 2 minutes at 11,000 rpm
  15. Discarded the flowthrough into waste receptacle
  16. Applied 0.5 mL of Buffer PB to spin column
  17. Centrifuged for additional 2 minutes at 11,000 rpm
  18. Discarded the flowthrough into waste receptacle
  19. Applied 0.75 mL of Buffer PE to spin column
  20. Centrifuged samples for additional 2 minutes at 11,000 rpm
  21. Discarded the flowthrough into waste receptacle
  22. Centrifuged for additional 2 minutes at 11,000 rpm until all residual fluid was removed from column
  23. Placed spin columns into second microcentrifuge tubes
  24. Added 50 μL of Buffer EB to columns
  25. Incubated samples at room temperature for at least 1 minute
  26. Centrifuged samples for additional 2 minutes at 11,000 rpm
  27. Placed samples into microcentrifuge rack for storage
• Note 1: Due to limitations, sample C was not used.
• Note 2: Due to limitations, procedure was conducted until step 12.
• April 4, 2012 (End time: 11:05)

• April 5, 2012 (Start time: 13:05)
• Procedure Part 1 (Start: 13:05):
  1. Combined .4g of Agarose with 50mL of TAE buffer to produce .8% agarose gel
  2. Designated the two A caps A1 and A2
  3. Designated the two B caps B1 and B2
  4. Prepared 4 additional caps pre-labeled A1, A2, B1 and B2
  5. Transferred 10 μL of each sample into respective labeled caps
  6. Added 2 μL of 1.5% Bromophenol Blue into each test cap
  7. Placed 12 μL of sample A1 in lane 2 of electrophoresis
  8. Placed 12 μL of sample B1 in lane 3 of electrophoresis
  9. Placed 12 μL of sample A2 in lane 4 of electrophoresis
  10. Placed 12 μL of sample B2 in lane 5 of electrophoresis
  11. Placed 1 Kb marker in lane 6 of electrophoresis
  12. Ran gel electrophoresis at 110V and .60A
• Procedure Part 1 (End: 13:50)
• Procedure Part 2 (Start: 16:05):
  1. Created 4 10 μL of buffer for samples A1, A2, B1, and B2 respectively
  2. For each, added 1 μL Buffer H, 1 μL BSA, 7.5 μL DNA, two different .5 μL enzymes with total of 1.0 μL
  3. Incubated the buffer at 37C for 1 hour
  4. Incubated the buffer at 65C for 15 minutes
• Procedure Part 2 (End: 16:50)
• April 5, 2012 (End time: 16:50)

• April 6, 2012 (Start time: 13:05)
• Sample name as follows:
  1. Sample 1: A1 and B1
  2. Sample 2: A1 and B2
  3. Sample 3: A2 and B1 Note: Sample 3 was disregarded due to the lack of B1)
  4. Sample 4: A2 and B2
• Procedure:
  1. Labeled separate test caps with sample numbers designated above
  2. Added 1 μL Buffer to each
  3. Added 1 μL Restriction Enzyme-H2O molecule complex (ratio is 2.5:2.5)
  4. Added 4 μL of sample category 'A'
  5. Added 4 μL of sample category 'B'
  6. Incubated at 37C overnight
• April 6, 2012 (End time: 13:40)

• April 9, 2012 (Start time: 13:15)
• Procedure:
  1. Designated 4 test tubes, labeled 1, 2, and 4 respectively
  2. Obtained chemically competent bacteria (to be later transformed)
  3. Added 10 ng (i.e. 10 μL) of plasmid
  4. Incubated the solution on ice for 10 minutes
  5. Heat shocked the solution at 42C for exactly 50 seconds
  6. Placed the samples on ice for 2 minutes
  7. Transfered bacteria-DNA mixture to 150 μL of enriched broth (LB + 5mM Glucose)
  8. Incubated the samples at room temperature for 45 minutes
  9. Incubated the samples at 37C overnight
• April 9, 2012 (End time: 14:50)

• April 10, 2012 (Start time: 12:15)
• Procedure:
  1. Drew grid structure on new plate
  2. Each cell of grid had arbitrary sample
  3. Labeled the grid structure
  4. Designated 11 test tubes
  5. Labeled each test tube with corresponding cell
  6. Streaked and grew 5 colonies from sample 1
  7. Streaked and grew 5 colonies from sample 2
  8. Streaked and grew 6 colonies from sample 4
• April 10, 2012 (End time: 12:45)

• April 11, 2012 (Start time: 13:10)
• Observation: Most of the colonies streaked were pink/red colored-signified not desired combination of plasmid.
• From observation, decided to streak additional colonies in search for desired plasmid combination.
• Procedure:
  1. Drew grid pattern on new plate
  2. Labeled columns A to F
  3. Designated columns A and B for sample 1
  4. Designated columns C and D for sample 2
  5. Designated columns E and F for sample 3
  6. Streaked colonies that appeared white on respective columns
  7. Incubated the samples at 37C overnight
• April 11, 2012 (End time: 13:35)

• April 12, 2012 (Start time: 13:20)
• Procedure:
  1. Examined the streaked bacteria culture: all of the cultures were red
  2. Concluded that the plasmid was not desired combination
  3. Planned for second method in determining desired plasmid combination
• April 12, 2012 (End time: 13:25)

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