IGEM:MIT/2007/Protocols

General Lab Protocols

• [[../Notebook/2007-6-11#General_Lab_Do_s_and_Don’t_s|General Lab Do's and Don'ts]]
• [[../Notebook/2007-6-13#Plating_on_Agar|Plating on Agar]]

DNA Work

• [[../Notebook/2007-6-13#Ligation|Ligation]]
• [[../Notebook/2007-6-13#Purifying_the_DNA|Purifying the DNA]]

Plasmid DNA Purification (microcentrifuge)

• 1. Harvest the bacterial cells by centrifugation at 5000rpm for 5 min. at 4˚C
• 2. Pipette/Decant out LB suspension
• 3. Resuspend pelleted bacterial cells in 0.3 ml Buffer P1 (in Falcone fridge) and transfer to a microcentrifuge tube
  • • • • • • • • • • ensure that RNase A has been added to Buffer P1
• 4. Add 0.3 ml of Buffer P2, mix thoroughly by vigorously inverting the sealed tube 4-6 times (or until homogenous), and incubate at room temperature for (no more than) 5 min.
  • • • • • • • • • • do not vortex
                    • close Buffer P2 immediately after use
• 5. Add 0.4 ml of chilled Buffer N3, mix immediately and thoroughly by vigorously inverting 4-6 times, and incubate (on ice) for 5 min.
• 6. Centrifuge for 10min. at 13,000 rpm
• 7. Apply the supernatants from step 4 into the QIAprep spin column by decanding or pipetting.
• 8. Centrifuge for 30-60 seconds on max speed. Discard flow-through
• 9. Wash QIAprep spin column by adding 0.5 ml Buffer PB and centrifuging for 30-60 s. Discard the flow-through.
• 10. Discard the flow-through, and centrifuge for an additional 1 min to remove residual wash buffer.
• 11. Place the QIAprep column in a clean 1.5 ml microcentrifuge tube. To elute DNA, add 50 ul water (or Buffer EB) to the center of each QIAprep spin column, let stand for 1 min, and centrifuge for 1 min.

Calculating Optical Density

Nanodrop Machinery

1. Start ND-1000 program
2. Select "nucleic acid"
3. 2 µL water
4. Push OK
5. Wipe top and bottom
6. 2 µL EB buffer (or water if used for elution)
7. Select "blank"
8. Wipe top and bottom
9. 2 µl sample
10. Select "measure"
11. Record the content of DNA in the lower right hand corner in ng/µl

PCR

1. Assemble 2 reaction tubes; one with a complete reaction and another without template that serves as a control
2. Set Reaction Tubes/Plates on Ice
3. Add the following components in a reaction vessel. Total volume should be between .5-20 µl.
   • 45 µl Platinum PCR SuperMix
   • Primers (200 nM final concentration per primer is recommended)
   • Template DNA solution
4. Cap reaction vessel and load into a thermal cycler at 94°C
5. Incubate tubes in a thermal cycler at 94°C for 30 s to 2 min
6. Perform the following PCR amplification
   1. 94°C for 4 minutes
   2. 94°C for 1 minute
   3. 55°C for 1 minute
   4. 72°C for 1 minute
   5. repeat steps 2-4 35 times
   6. 72°C for 10 minutes
   7. 4°C forever

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

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 (37ºC) 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

Making stock solution of 1% Agarose in TAE for Gel electrophoresis

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
   1. 1 mg/mL = 100% [water]
   2. 10 mg/mL = 1%
7. Get agarose and weigh out 10 mg using boat or paper
8. Pour agarose into large flask
9. Clean up weighing area (brush)
10. Get 1 L graduated cylinder (not sterile) and fill with 1x TAE buffer
11. Pour in half the TAE buffer from the cylinder into the flask and put in microwave
12. Wait until bubbling and mix/stir – continue pouring in more buffer until clear
13. Store in 55ºC box with cap halfway on (no explosions)