Paulsson:Protocols: Difference between revisions

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===Agarose Gels===
===Agarose Gels===
For small casting tray, 0.6g agarose + 60 ml TAE, microwave 55 sec, add 3ul EthBr stock, let stand for 8 min before pouring<Br>
*For small casting tray, 0.6g agarose + 60 ml TAE, microwave 50-60 sec, add 3ul EthBr stock, let stand for 7 min before pouring<Br>
Narrow wells: 1.5 x 2.59mm, ~19ul volume<br>
Narrow wells (15 lanes): 1.5 x 2.59mm, ~19ul volume<br>
Wide wells: 1.5 x 5.54mm, ~40ul volume  (depending on thickness of gel, of course)
Wide wells (8 lanes): 1.5 x 5.54mm, ~40ul volume   


For large casting tray, 150ml volume, microwave
*For large casting tray, 150ml volume, microwave 80-90 sec, add 7.5ul EthBr stock, let stand 10min before pouring<Br>
Narrow wells (20 lanes): 1.5 x ##mm, ~##ul volume<br>
Wide wells ( lanes): 1.5 x ##mm, ~##ul volume <br>
Multi-channel pipet compatible wells (26 lanes): 1.5 x mm, ~##ul volume <br>

Revision as of 16:40, 24 January 2008

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Many excellent protocols can be found in Current Protocols in Molecular Biology, available through the Countway Digital Library.

Please contribute to this page!!!

Preparation of electrocompetent E.coli
Electroporation of E.coli
Measuring DNA replication with 3H-thymidine

Nanodrop
Plate Reader

Getting Started In the Lab

Pipetting 101

general
what volume, which tool
accuracy and calibration (cleaning)

Sterile technique

Nurturing Microbes

preservation growth conditions genotypes antibiotics

Media Preparation

Current Protocols in Molecular Biology article on mimimal, rich, solid and agar media for E. coli
Media:Ecoli_preparation_media.pdf

  • MSC media (from Rosenfeld et al., Science v307, 25 March 2005, SOM)

M9 salts
0.6% succinate
0.01% casamino acids
0.15 ug/ml biotin
1.5 uM thiamine
doubling time for E. coli reported to be 45 (-/+10) min at 32 degrees
for microscope pad, dissolve low melt agarose in MSC (1.5%)

Plasmid Biology

definitions transformation isolation segragation incompatability copy number selection


Manipulating DNA

Synthesis PCR Enzymes
Quick Change mutagenesis
Colony PCR

Agarose Gels

  • For small casting tray, 0.6g agarose + 60 ml TAE, microwave 50-60 sec, add 3ul EthBr stock, let stand for 7 min before pouring

Narrow wells (15 lanes): 1.5 x 2.59mm, ~19ul volume
Wide wells (8 lanes): 1.5 x 5.54mm, ~40ul volume

  • For large casting tray, 150ml volume, microwave 80-90 sec, add 7.5ul EthBr stock, let stand 10min before pouring

Narrow wells (20 lanes): 1.5 x ##mm, ~##ul volume
Wide wells ( lanes): 1.5 x ##mm, ~##ul volume
Multi-channel pipet compatible wells (26 lanes): 1.5 x mm, ~##ul volume