DIYbio/BOSSlab/Notebook/2011/05/11

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|style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;"> Project name</span>
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|style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;"> BOSSLAB</span>
|style="background-color: #F2F2F2" align="center"|<html><img src="/images/9/94/Report.png" border="0" /></html> [[{{#sub:{{FULLPAGENAME}}|0|-11}}|Main project page]]<br />{{#if:{{#lnpreventry:{{FULLPAGENAME}}}}|<html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>[[{{#lnpreventry:{{FULLPAGENAME}}}}{{!}}Previous entry]]<html>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</html>}}{{#if:{{#lnnextentry:{{FULLPAGENAME}}}}|[[{{#lnnextentry:{{FULLPAGENAME}}}}{{!}}Next entry]]<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html>}}
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==Preflight==
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== Preflight ==
=== what's our goal ===
=== what's our goal ===
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* hot water; combusted propane gas
* hot water; combusted propane gas
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=== Protocol ===
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== Protocol ==
# Thaw competent cells (Lyocomp E. coli) on ice.
# Thaw competent cells (Lyocomp E. coli) on ice.
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# Incubate plates at 37°C for 18 h.  
# Incubate plates at 37°C for 18 h.  
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== Visual Protocol ==
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<a href="http://www.flickr.com/photos/macowell/5713410863/" title="2011-05-11 visual protocol by macowell, on Flickr"><img src="http://farm4.static.flickr.com/3421/5713410863_fc62439a0e.jpg" width="500" height="374" alt="2011-05-11 visual protocol"></a>
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Current revision

BOSSLAB Main project page
Next entry

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

  1. Thaw competent cells (Lyocomp E. coli) on ice.
  2. During this time pre-chill sterile centrifuge tubes on ice.
  3. Gently tap competent cell containers to mix
  4. Aliquot bacterial suspension in pre-chilled tubes (100 µl per tube).
  5. To the aliquots add 1 µl of plasmid (RFP PUC19).
  6. Gently tap centrifuge tubes to mix
  7. Incubate 30 min on ice.
  8. During this time pre-heat water bath to 42°C (don’t use heating block).
  9. Incubate samples at 42°C in the water bath for 60s. Do not shake.
  10. Immediately chill samples on ice for 2 minutes.
  11. Add 0.95 ml of room temperature SOC.
  12. Plate 10 µl of bacterial suspension on antibiotic-containing agar plate. (filter sterilized Kanamycin100 ug/ml?).
  13. Transfer some bacteria to another antibiotic-containing plate using a loop.
  14. Incubate plates at 37°C for 18 h.

Visual Protocol

2011-05-11 visual protocol

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