Difference between revisions of "User:Matthewmeisel"

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(Mon Jun 12: transformation)
m (Wed Jun 14)
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** 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
 
** 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
 
** E0241 digested with XbaI and PstI in order to cleave it as a fragment
 +
* procedure:
 
#The following ingredients were each added to four 1.5 ml centrifuge tubes:
 
#The following ingredients were each added to four 1.5 ml centrifuge tubes:
 
#*10 μl water
 
#*10 μl water

Revision as of 12:05, 14 June 2006

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:
  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
  • procedure:
  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.

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 (upload gel image)

Transformation of standard components

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)
  • procedure:
  1. -->procedure here!<--
  2. Plated the mixture on agar plates and incubated at 37°C for 24 h.

Bibliography

  1. 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 | PubMed ID:14961116 | HubMed [shih0]
  1. 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 | PubMed ID:11278776 | HubMed [shih1]
  1. 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. PubMed ID:11007486 | HubMed [shih2]


Other projects

My work in Biophysics 101

Contact info

Email me: (my last name) at fas dot harvard dot edu