IGEM:MIT/2007/Protocols: Difference between revisions
m (→Purification) |
|||
Line 1: | Line 1: | ||
=== 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 | |||
3. 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 | |||
4. 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. | |||
5. Centrifuge for 10min. at 13,000 rpm | |||
6. Apply the supernatants from step 4 into the QIAprep spin column by decanding or pipetting. | |||
7. Centrifuge for 30-60 seconds on max speed. Discard flow-through | |||
8. Wash QIAprep spin column by adding 0.5 ml Buffer PB and centrifuging for 30-60 s. Discard the flow-through. | |||
9. Discard the flow-through, and centrifuge for an additional 1 min to remove residual wash buffer. | |||
10. 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 === | === Calculating Optical Density === | ||
Nanodrop Machinery | Nanodrop Machinery |
Revision as of 11:12, 12 June 2007
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 3. 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 4. 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. 5. Centrifuge for 10min. at 13,000 rpm 6. Apply the supernatants from step 4 into the QIAprep spin column by decanding or pipetting. 7. Centrifuge for 30-60 seconds on max speed. Discard flow-through 8. Wash QIAprep spin column by adding 0.5 ml Buffer PB and centrifuging for 30-60 s. Discard the flow-through. 9. Discard the flow-through, and centrifuge for an additional 1 min to remove residual wash buffer. 10. 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
- Start ND-1000 program
- Select "nucleic acid"
- 2 µL water
- Push OK
- Wipe top and bottom
- 2 µL EB buffer (or water if used for elution)
- Select "blank"
- Wipe top and bottom
- 2 µl sample
- Select "measure"
- Record the content of DNA in the lower right hand corner in ng/µl
PCR
- Assemble 2 reaction tubes; one with a complete reaction and another without template that serves as a control
- Set Reaction Tubes/Plates on Ice
- 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
- Cap reaction vessel and load into a thermal cycler at 94°C
- Incubate tubes in a thermal cycler at 94°C for 30 s to 2 min
- Perform the following PCR amplification
- 94°C for 4 minutes
- 94°C for 1 minute
- 55°C for 1 minute
- 72°C for 1 minute
- repeat steps 2-4 35 times
- 72°C for 10 minutes
- 4°C forever
Purification
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)