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Week 1

Thu Jun 15

Transformation of standard components

  • ligation and transformation of promoter and GFP
  • ligation protocol with the Roche Rapid DNA ligation kit:
  1. 6 μL of insert (E0241), 1 μL of plasmid (R0010 and backbone), and 3 μL of 1x DNA dilution buffer (vial 2) (enough buffer for total volume of 10 μL) into a microcentrifuge tube. (Would ordinarily use 2 μL of plasmid, but the plasmid concentration is about doubled because it combined bands from two gel lanes.)
  2. Mixed ligation buffer (vial 1), and added 10 μL to tube.
  3. Added 1 μL T4 DNA ligase (vial 3).
  4. Incubated on ice for 5 min.
  • transformation:
  1. 30 μL competent cells each into three microcentrifuge tubes.
  2. Ligation mixture, 1 μL water (negative control), and 1 μL pUC 19 (positive control) added to respective tubes.
  3. Incubated the tubes on ice for 20 min.
  4. Heat shocked the tubes at 42°C for 30 s.
  5. Added 200 μL SOC to each tube.
  6. Incubated, with shaking, at 37°C for 1 h.
  7. Plated the mixture on ampicillin plates and incubated at 37°C for 24 h.

Wed Jun 14

Transformation of standard components

  • cultured bacteria did grow overnight
  • 1 mL of each tube reserved to make glycerol stock
  • extracted DNA from remaining 4 mL using the standard protocol:
  1. Resuspended pelleted bacterial cells in 250 µl Buffer P1 (w/ RNAse) (kept at 4 °C) and transfered to a microcentrifuge tube.
  2. Added 250 μl Buffer P2 and gently inverted the tube 4–6 times to mix. Waited 2 min.
  3. Added 350 μl Buffer N3 and inverted the tube immediately but gently 4–6 times. Solution became cloudy.
  4. Centrifuged for 10 min at 14,000 rpm. A compact white pellet formed.
  5. Applied the supernatants from step 4 to the QIAprep spin column by decanting.
  6. Centrifuged for 60 s. Discarded the flow-through.
  7. Washed the QIAprep spin column with 0.5 ml Buffer PB and centrifuged for 60 s. Discarded the flow-through.
  8. Washed QIAprep spin column by with 0.75 ml Buffer PE and centrifuged for 60 s.
  9. Placed the QIAprep column in a clean 1.5 ml microcentrifuge tube. Eluted with 30 μl water to the center of each QIAprep spin column, let stand for 2 min, and centrifuged for 60 s.
  • digested promoter (R0010) and GFP (E0241) plasmids
    • R0010 digested with SpeI and PstI in order to leave it attached at upstream end to the plasmid backbone (otherwise, the fragment would only be ~200 bp long, which is a little too short for electrophoresis with much longer fragments
    • E0241 digested with XbaI and PstI in order to cleave it as a fragment
  1. The following ingredients were each added to four 1.5 ml centrifuge tubes:
    • 10 μl water
    • 8 μl DNA from the previous step (R0010-1, R0010-2, E0241-1, E0241-2 in respective tubes)
    • 2.5 μl 10x NEB buffer (#2 for R0010-01 and R0010-2, #3 for E0241-1 and E0241-2)
    • 2.5 μl 10x BSE
    • 1.0 μl 1:1 diluted enzyme A (SpeI for R0010-1 and R0010-2, XbaI for E0241-1 and E0241-2)
    • 1.0 μl 1:1 diluted enzyme B (PstI for all).
  2. Tubes were incubated at 37°C for 90 min.
  3. Tubes were incubated at 80°C for 15 min in order to inactivate the enzymes.
  4. All four samples were run on a 1% agarose gel:
Lane Contents Loading Buffer
01kb DNA ladder (10 μL)10x loading dye (1.1 μL)
1E0241-1 (15 μL)10x loading dye (2.5 μL)
2E0241-2 (15 μL)10x loading dye (2.5 μL)
3R0010-1 (15 μL)10x loading dye (2.5 μL)
4R0010-2 (15 μL)10x loading dye (2.5 μL)

Tue Jun 13

DNA nanostructure reaction PCR

  • order of lanes: 1) 1kb ladder, 2) full rxn (oligos + scaffold), 3) just scaffold, 4) just oligos
  • run on 2% agarose gel
  • appears that reaction was successful: gel image (leftmost four lanes) shows expected results
    • assembled nanostructure runs slightly faster than scaffold, oligos appear as a smear of short DNAs

Transformation of standard components

  • results of plated bacteria: all three plates showed colony growth, negative control did not
  • colony selection and amplification
  1. Seven culture tubes were filled with 5 mL LB medium and 40 μl (??) (?x) ampicillin
  2. Colonies were selected from the three plates (three from R0010, two from E7104, two from E0241) and were transferred into respective culture tubes with a sterilized pick
  3. Culture tubes were incubated, with shaking, at 37°C for 18 h.
  4. Plates were stored at 4°C.

Mon Jun 12

DNA nanostructure reaction PCR

Transformation of standard components

  • goal: insert three BioBrick plasmids (already containing BioBricks) into E. coli in order to amplify them
    • a positive control (E7104) with the T7 promoter upstream of GFP
    • the lac operon (R0010)
    • GFP (E0241)
  • transformation protocol:
  1. 1 μL each of E7104, R0010, E0241, and water into separate microcentrifuge tubes that each contain 30 μL of competent cells
  2. Incubated the tubes on ice for 20 min.
  3. Heat shocked the tubes at 42°C for 30 s.
  4. Added 200 μL SOC to each tube.
  5. Incubated, with shaking, at 37°C for 1 h.
  6. Plated the mixture on ampicillin plates and incubated at 37°C for 24 h.


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