Difference between revisions of "User:Matthewmeisel"

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(Mon Jun 12: details of transformation)
m (Wed Jun 14)
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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]], eluted with water
+
* 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 &mu;l 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:
  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. Discard 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.</pre>

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 plasmids (already containing BioBricks) into /E. coli/ in order to amplify them

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