User:Matthewmeisel
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Quick link: iGEM:Harvard/2006
Notebook
Week 1
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:
- Resuspended pelleted bacterial cells in 250 µl Buffer P1 (w/ RNAse) (kept at 4 °C) and transfered to a microcentrifuge tube.
- Added 250 μl Buffer P2 and gently inverted the tube 4–6 times to mix. Waited 2 min.
- Added 350 μl Buffer N3 and inverted the tube immediately but gently 4–6 times. Solution became cloudy.
- Centrifuged for 10 min at 14,000 rpm. A compact white pellet formed.
- Applied the supernatants from step 4 to the QIAprep spin column by decanting.
- Centrifuged for 60 s. Discarded the flow-through.
- Washed the QIAprep spin column with 0.5 ml Buffer PB and centrifuged for 60 s. Discarded the flow-through.
- Washed QIAprep spin column by with 0.75 ml Buffer PE and centrifuged for 60 s.
- 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
- 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).
- Tubes were incubated at 37°C for 90 min.
- Tubes were incubated at 80°C for 15 min in order to inactivate the enzymes.
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
- Seven culture tubes were filled with 5 mL LB medium and 40 μl (??) (?x) ampicillin
- 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
- Culture tubes were incubated, with shaking, at 37°C for 18 h.
- Plates were stored at 4°C.
Mon Jun 12
DNA nanostructure reaction PCR
- standard protocol with materials from Shawn
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)
- -->procedure here!<--
- Plated the mixture on agar plates and incubated at 37°C for 24 h.
Bibliography
- Shih WM, Quispe JD, and Joyce GF. A 1.7-kilobase single-stranded DNA that folds into a nanoscale octahedron. Nature. 2004 Feb 12;427(6975):618-21. DOI:10.1038/nature02307 |
- Shih WM and Spudich JA. The myosin relay helix to converter interface remains intact throughout the actomyosin ATPase cycle. J Biol Chem. 2001 Jun 1;276(22):19491-4. DOI:10.1074/jbc.M010887200 |
- Shih WM, Gryczynski Z, Lakowicz JR, and Spudich JA. A FRET-based sensor reveals large ATP hydrolysis-induced conformational changes and three distinct states of the molecular motor myosin. Cell. 2000 Sep 1;102(5):683-94. DOI:10.1016/s0092-8674(00)00090-8 |
Other projects
My work in Biophysics 101
Contact info
Email me: (my last name) at fas dot harvard dot edu