User:Matthewmeisel

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

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

• 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

• 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)

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 |

• Harvard link

2. 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 |

• Harvard link

3. 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 |

• Harvard link

Other projects

My work in Biophysics 101

Contact info

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