IGEM:MIT/2007/Notebook/2007-6-11

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(General Lab Do s and Don’t s)
(Transforming DNA plasmids into bacterial colonies on plates)
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*Must pick up colonies to grow – inoculation in test tubes – grow over night in media
*Must pick up colonies to grow – inoculation in test tubes – grow over night in media
*Put antibiotic in media – all non-plasmid containing bacteria will die (selective amplification)
*Put antibiotic in media – all non-plasmid containing bacteria will die (selective amplification)
-
*Antibiotic into LB:  500 LB à 500 microliters of Amp
+
*Antibiotic into LB:  500 LB needs 500 microliters of Amp
*Use Bunsen burner, gloves
*Use Bunsen burner, gloves
*500 microliters of Amp into LB flask after warming near lip of LB bottle  
*500 microliters of Amp into LB flask after warming near lip of LB bottle  
Line 58: Line 58:
*Touch metal loop lightly to one colony and touch to the top of LB broth; mix in broth
*Touch metal loop lightly to one colony and touch to the top of LB broth; mix in broth
*A tip:  Distinguish before/after inoculation tubes by placing in different rows of the test tube holder
*A tip:  Distinguish before/after inoculation tubes by placing in different rows of the test tube holder
-
*Put the test tubes into incubator in warm (37 degree) room – make sure you switch off the rotating device first, put in all tubes, and then TURN BACK ON!
+
*Put the test tubes into incubator in warm (37ºC) room – make sure you switch off the rotating device first, put in all tubes, and then TURN BACK ON!
*Note the time that incubation starts (for today, 16.29)
*Note the time that incubation starts (for today, 16.29)
*If something is growing in culture that should not be there, dump it out after pouring bleach inside
*If something is growing in culture that should not be there, dump it out after pouring bleach inside
*Clean up: Parafilm all containers
*Clean up: Parafilm all containers
*LB and Amp go in fridge – must warm up before using next time
*LB and Amp go in fridge – must warm up before using next time
-
 
===Making Plates===
===Making Plates===

Revision as of 23:11, 11 June 2007

Contents

Day Outline

  • 9am: Meet at 68-577
  • Morning: Primer design (BE 109 protocol)
  • Afternoon: Topic/Paper research

LAB WORK

please fill in details

  • added Amp to 500ml LB
  • innoculate 5ml LB/Amp with bact. colonies (EGFP and NNX)
  • made 1 liter 1% agarose solution

....................

Enjoy the tips and protocols below -- and add any suggestions if you'd like! --SK

General Lab Do s and Don’t s

  • Pipettes: don’t use over maximum amount/under minimum amount; don’t contaminate pipette by using higher than necessary
    • Different pipette tips exist – look at the labels [filters, sizes, etc.]
    • When pipetting up, get used to knowing where fluids should end
    • Pipetting fluid – make sure suck up full volume (don’t take out of container too quickly); don’t touch bottom of container; push pipette down first and then pull up (don’t make bubbles)
    • Dispensing – go down both notches
    • Sucking up – go down one notch
    • Resuspension – mixing by continually sucking up and down (NO BUBBLES)
    • Look at different numbers on pipettes
    • If desired, practice on cup of water!
  • KNOW HOW MUCH ONE MICROLITER IS (for PCR)
  • Put all enzymes inside cooling blocks next to freezers to avoid denaturation [such as Amp for LB]
  • Stockroom is also used for carcinogens – DNA staining chemical – don’t touch anything!
  • Before work: spray ethanol on bench and gloves; Kimwipe (not on open flame!)
  • Be as sterile as possible! LB is made in building 68 – can pick up more
  • Automatic Bunsen burners – continuous or sensor
  • Autoclaved water is sterile

Transforming DNA plasmids into bacterial colonies on plates

  • Must pick up colonies to grow – inoculation in test tubes – grow over night in media
  • Put antibiotic in media – all non-plasmid containing bacteria will die (selective amplification)
  • Antibiotic into LB: 500 LB needs 500 microliters of Amp
  • Use Bunsen burner, gloves
  • 500 microliters of Amp into LB flask after warming near lip of LB bottle
  • Discard tips into small buckets on top of bench; dump into red sharps container at end of day
  • Mark remaining Amp volume with marker
  • Freezer for iGEM: white, shared for now; later will have freestanding under bench
  • Use lab tape to re-label and update flasks/containers with substance name, iGEM, date and your initials
  • Plates go into metal container [opposite bench]
  • Keep LB as sterile as possible by opening only once – pour needed amount into sterile blue screw cap bottles and label with name, iGEM, date and initials
  • Transferring LB to blue screw cap – lid of LB down on bench; put LB bottle over Bunsen burner
  • Temporary fridge space in front of bench
  • Bacteria with two different types of vectors on streaked plates – make far enough apart to form colony
  • Make sure automatic pipetter is charging; don’t suck up into device
  • 70% ethanol mix: to 400 with ethanol; rest with distilled water
  • Glass test tubes of two plasmids – label with name of plasmid and all other relevant details – both cap and tube
  • Use 10 mL pipette (glass) and automatic pipette (upper button is up) – 5 mL of LB Amp into each tube (inoculation of 5 mL)
  • Don’t open glass tube cap until necessary
  • Glass pipettes into liquid-containing plastic waste jar
  • Flame the metal loop
  • Use two fingers to lift cap of glass tube
  • Flame top of tube before and after touching bacterial colony
  • Let metal loop cool to temperature of gel by stabbing loop on agar with no colonies
  • Touch metal loop lightly to one colony and touch to the top of LB broth; mix in broth
  • A tip: Distinguish before/after inoculation tubes by placing in different rows of the test tube holder
  • Put the test tubes into incubator in warm (37ºC) room – make sure you switch off the rotating device first, put in all tubes, and then TURN BACK ON!
  • Note the time that incubation starts (for today, 16.29)
  • If something is growing in culture that should not be there, dump it out after pouring bleach inside
  • Clean up: Parafilm all containers
  • LB and Amp go in fridge – must warm up before using next time

Making Plates

  • 1% agarose gel
  • stock agarose solution (for gels)
  • 1 Liter flask
  • Anything with foil on it is sterile – LB flask does not have to be sterile, since it will be heated anyway
  • Label 1% agarose, iGEM, TAE buffer, date
  • 1% is weight per volume
    • 1 mg/mL = 100% [water]
    • 10 mg/mL = 1%
  • Get agarose and weigh out 10 mg using boat or paper
  • Pour agarose into large flask
  • Clean up weighing area (brush)
  • Get 1 L graduated cylinder (not sterile) and fill with 1x TAE buffer
  • Pour in half the TAE buffer from the cylinder into the flask and put in microwave
  • Wait until bubbling and mix/stir – continue pouring in more buffer until clear
  • Store in 55ºC box with cap halfway on (no explosions)

POSSIBLE IDEAS

Leading Concepts

Illuminate in Dark- Maybe

Sticky Bacteria- lacking application but maybe

Requiring Further Research

Synthesizing Vitamins – Further research TTP JCH

Extreme environment – yes LBH SK

Chitin Bandaid- More info AWL

FOOD? Spoilage/Binding to plastic – yes AGK

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