IGEM:MIT/2007/Protocols

Protocols

PCR

Platinum PCR Supermix

1. Set Reaction Tubes/Plates on Ice
2. Add the following components in a reaction vessel

• • 45 µl Platinum PCR SuperMix
  • Primers (200 nM final concentration per primer is recommended)
  • Template DNA solution

Total volume should be between .5-20 µl.

1. Cap reaction vessel and load into a thermal cycler at 94°C
2. Incubate tubes in a thermal cycler at 94°C for 30 s to 2 min
3. Perform 25-35 cycles of PCR amplification
   1. Denature 94° for 15-30 s
   2. Anneal 55°C for 15-30 s
   3. Extend 72°C for 1 min per kb

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

Growing up bacterial colonies from 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 stock solution of 1% Agarose in TAE for Gel electrophoresis

• 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)