User:Matthewmeisel: Difference between revisions
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* cultured bacteria did grow overnight | * cultured bacteria did grow overnight | ||
* 1 mL of each tube reserved to make glycerol stock | * 1 mL of each tube reserved to make glycerol stock | ||
* extracted DNA from remaining 4 mL using [[Miniprep/Qiagen_kit|standard protocol]], | * extracted DNA from remaining 4 mL using the [[Miniprep/Qiagen_kit|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. Discard 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.</pre> | |||
====Tue Jun 13==== | ====Tue Jun 13==== | ||
Revision as of 09:41, 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:
- 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. Discard 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.
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
- standard protocol with materials from Shawn
Transformation of standard components
- goal: insert three plasmids (already containing BioBricks) into /E. coli/ in order to amplify them
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
