DIYbio/BOSSlab/Notebook/2011/05/15: Difference between revisions
From OpenWetWare
Jump to navigationJump to search
(Autocreate 2011/05/15 Entry for DIYbio/BOSSlab/Notebook) |
|||
Line 6: | Line 6: | ||
| colspan="2"| | | colspan="2"| | ||
<!-- ##### DO NOT edit above this line unless you know what you are doing. ##### --> | <!-- ##### DO NOT edit above this line unless you know what you are doing. ##### --> | ||
== | == Preflight == | ||
* | what's our goal: | ||
transformation of Invivogen lyocomp cells w/ BBa_J04450 on card (circa 2008). | |||
=== Materials === | |||
* lyocomp E. coli 116, (k12 derived?), non-pathogenic. http://www.invivogen.com/family.php?ID=189 | |||
* BBa_J04450 plasmid-on-card. Ampicillin resistant, based on pSB1A4. http://partsregistry.org/Part:pSB4A1 | |||
* 1000x stock of ampicillin | |||
* LB-agar (1 L) | |||
* SOC, 1.8 mL | |||
* pressure sterilizer, burner, propane | |||
* ice, water, 50g scale, fridge, freezer, incubator, p200, p1000, tips, gloves, bleach | |||
=== what are the risks to us === | |||
* fire hazard: pressure sterilizer + propane burner | |||
* chemical hazard: bleach | |||
* chemical hazard: concentrated ampicillin | |||
* experimental hazard: lab-grade E. coli contamination on bench surfaces | |||
* temperature hazard: molten LB-agar | |||
=== what waste are we gonna generate === | |||
* bleach + LB-agar (solid) | |||
* combusted propane gas | |||
* water | |||
* tips used to transfer & mix transformed E. coli (bleached) | |||
=== mitigation of community risks === | |||
* chemical hazards: propane leak in air | |||
** only use burner outdoors on concrete | |||
* e. coli on tips | |||
** dispose of pipet tips into bleach solution, bag & throw away. | |||
== Protocol == | |||
based on http://www.invivogen.com/PDF/LyoComp_GT116_TDS.pdf | |||
# Thaw competent cells (Lyocomp E. coli 116) & rehydration solution on ice. | |||
# Chill 1.5 mL centrifuge tubes on ice. | |||
# Rehydrate lyophilized cells with 0.5 mL "rehydration solution" | |||
# Let cells rehydrate for 30 min on ice | |||
# Add "DNA chads" from biobrick card to 1.5 mL centrifuge tubes | |||
# dispense 250 mL of rehydrated lyocomp cells to each 1.5 mL centrifuge tube. 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 exactly 30s. | |||
# Immediately chill samples on ice for 2 minutes. | |||
# Add 900 uL of room temperature SOC to each tube. | |||
# Incubate at 37 C for 90 min, shaking at 250 RPM | |||
# Spread 100 uL of transformed bacteria on LB-Amp plates. | |||
# Incubate plates at 37°C for 18 h. | |||
== Notes == | |||
* p1000 tips don't fit in lyocomp vial. Used p200 tips. Only removed 400 uL (200 uL to each 1.5 mL tube) of rehydrated e. coli from the vial. | |||
* unsure of concentration and age of frozen Amp stock. Assuming 1000x. Poured two LB plates without the amp. Added 100 uL transformant to each. | |||
* distributed 100 uL transformant to two LB-Amp plates. | |||
* incubating one 1.5 mL tube with remaining transformant (900 uL) at 37 C. no shaking. | |||
* storing the other 1.5 mL tube with remaining transformant (900 uL) at 4 C. | |||
* incubation started at xxx PM 5/15/2011 | |||
Revision as of 19:17, 15 May 2011
Project name | <html><img src="/images/9/94/Report.png" border="0" /></html> Main project page <html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>Previous entry<html> </html> |
Preflightwhat's our goal: transformation of Invivogen lyocomp cells w/ BBa_J04450 on card (circa 2008). Materials
what are the risks to us
what waste are we gonna generate
mitigation of community risks
Protocolbased on http://www.invivogen.com/PDF/LyoComp_GT116_TDS.pdf
Notes
|