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

Day Outline

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

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

innoculate 5ml LB/Amp with bact. colonies (EGFP and NNX)

 * 1) Must pick up colonies to grow – inoculation in test tubes – grow over night in media
 * 2) Put antibiotic in media – all non-plasmid containing bacteria will die (selective amplification)
 * 3) Antibiotic into LB: 500 LB needs 500 microliters of Amp
 * 4) Use Bunsen burner, gloves
 * 5) 500 microliters of Amp into LB flask after warming near lip of LB bottle
 * 6) Discard tips into small buckets on top of bench; dump into red sharps container at end of day
 * 7) Mark remaining Amp volume with marker
 * 8) Freezer for iGEM: white, shared for now; later will have freestanding under bench
 * 9) Use lab tape to re-label and update flasks/containers with substance name, iGEM, date and your initials
 * 10) Plates go into metal container [opposite bench]
 * 11) 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
 * 12) Transferring LB to blue screw cap – lid of LB down on bench; put LB bottle over Bunsen burner
 * 13) Temporary fridge space in front of bench
 * 14) Bacteria with two different types of vectors on streaked plates – make far enough apart to form colony
 * 15) Make sure automatic pipetter is charging; don’t suck up into device
 * 16) 70% ethanol mix: to 400 with ethanol; rest with distilled water
 * 17) Glass test tubes of two plasmids – label with name of plasmid and all other relevant details – both cap and tube
 * 18) Use 10 mL pipette (glass) and automatic pipette (upper button is up) – 5 mL of LB Amp into each tube (inoculation of 5 mL)
 * 19) Don’t open glass tube cap until necessary
 * 20) Glass pipettes into liquid-containing plastic waste jar
 * 21) Flame the metal loop
 * 22) Use two fingers to lift cap of glass tube
 * 23) Flame top of tube before and after touching bacterial colony
 * 24) Let metal loop cool to temperature of gel by stabbing loop on agar with no colonies
 * 25) Touch metal loop lightly to one colony and touch to the top of LB broth; mix in broth
 * 26) A tip: Distinguish before/after inoculation tubes by placing in different rows of the test tube holder
 * 27) Put the test tubes into incubator in warm (37°C) room – make sure you switch off the rotating device first, put in all tubes, and then TURN BACK ON!
 * 28) Note the time that incubation starts (for today, 16.29)
 * 29) If something is growing in culture that should not be there, dump it out after pouring bleach inside
 * 30) Clean up: Parafilm all containers
 * 31) LB and Amp go in fridge – must warm up before using next time

made 1 liter 1% agarose solution

 * 1) 1% agarose gel
 * 2) Stock agarose solution (for gels)
 * 3) 1 Liter flask
 * 4) Anything with foil on it is sterile – LB flask does not have to be sterile, since it will be heated anyway
 * 5) Label 1% agarose, iGEM, TAE buffer, date
 * 6) 1% is weight per volume
 * 7) 1 mg/mL = 100% [water]
 * 8) .01 mg/mL = 1%
 * 9) Get agarose and weigh out 10 mg using boat or paper
 * 10) Pour agarose into large flask
 * 11) Clean up weighing area (brush)
 * 12) Get 1 L graduated cylinder (not sterile) and fill with 1x TAE buffer
 * 13) Pour in half the TAE buffer from the cylinder into the flask and put in microwave
 * 14) Wait until bubbling and mix/stir – continue pouring in more buffer until clear
 * 15) Store in 55°C box with cap halfway on (no explosions)

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