DIYbio/BOSSlab/Notebook/2011/05/11: Difference between revisions
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== | ==Preflight== | ||
* | |||
=== what's our goal === | |||
dry-run through transformation protocol w/ BBa_J04450 on card using Invivogen lyocomp cells | |||
* lyocomp 116 cells, e. coli k12 (?), non-pathogenic | |||
* hot water bath; 42C, heat caution | |||
* Ian: "The chemicals we'll be using amount to waste water (E. coli), cheese whey (tryptone), Vegemite (yeast extract & NaCl), tofu firming salts (MgCl2 and MgSO4), jelly (agar) and antibiotics and the concentrations are fairly low, so no special dangers - i.e. don't eat it. Cleanup can be city sewer, since all of these are things that are already found in waste water." | |||
=== what are the risks today to us === | |||
* trivial; just doing a dry run w/ the water baths | |||
=== what are the risks today to the community === | |||
* water | |||
=== what waste are we gonna generate === | |||
* hot water; combusted propane gas | |||
=== Protocol === | |||
# Thaw competent cells (Lyocomp E. coli) on ice. | |||
# During this time pre-chill sterile centrifuge tubes on ice. | |||
# Gently tap competent cell containers to mix | |||
# Aliquot bacterial suspension in pre-chilled tubes (100 µl per tube). | |||
# To the aliquots add 1 µl of plasmid (RFP PUC19). | |||
# Gently tap centrifuge tubes to mix | |||
# Incubate 30 min on ice. | |||
# During this time pre-heat water bath to 42°C (don’t use heating block). | |||
# Incubate samples at 42°C in the water bath for 60s. Do not shake. | |||
# Immediately chill samples on ice for 2 minutes. | |||
# Add 0.95 ml of room temperature SOC. | |||
# Plate 10 µl of bacterial suspension on antibiotic-containing agar plate. (filter sterilized Kanamycin100 ug/ml?). | |||
# Transfer some bacteria to another antibiotic-containing plate using a loop. | |||
# Incubate plates at 37°C for 18 h. | |||
Revision as of 11:53, 12 May 2011
Project name | <html><img src="/images/9/94/Report.png" border="0" /></html> Main project page Next entry<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html> |
Preflightwhat's our goaldry-run through transformation protocol w/ BBa_J04450 on card using Invivogen lyocomp cells
what are the risks today to us
what are the risks today to the community
what waste are we gonna generate
Protocol
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