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| === Plasmid DNA Purification (microcentrifuge) === | | ==General Lab Protocols== |
| | *[[../Notebook/2007-6-11#General_Lab_Do_s_and_Don’t_s|General Lab Do's and Don'ts]] |
| | *[[../Notebook/2007-6-12#Nanodrop (~20ng/ul) Protocol|Calculating Optical Density; Nanodrop Protocol]] |
| | *[[../Notebook/2007-6-12#Obtaining a Cast|Making a Cast]] |
| | *[[../Notebook/2007-6-11#made_1_liter_1%_agarose_solution|Making Agarose Solution]] |
| | *[[../Notebook/2007-6-12#Protocol for Preparing Agarose Gel|Making an Agarose Gel]] |
| | *[[../Notebook/2007-6-12#Protocol for Preparing to Run a Gel Electrophoresis|Preparing a Gel Electrophoresis]] |
| | *[[../Notebook/2007-6-12#Protocol for Running a Gel Electrophoresis|Running a Gel Electrophoresis]] |
| | *[[../Notebook/2007-6-13#Making_LB_Amp_Agar_Plates|Making LB Amp Agar Plates]] |
| | *[[../Notebook/2007-6-11#innoculate_5ml_LB/Amp_with_bact._colonies_(EGFP_and_NNX)|Innoculating LB with bacteria]] |
| | *[[../Notebook/2007-6-12#Protocol for Imaging Result and Gel Diagnostic|Gel Imaging]] |
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| 1. Harvest the bacterial cells by centrifugation at 5000rpm for 5 min. at 4˚C
| | ==DNA Work== |
| 2. Pipette/Decant out LB suspension
| | *[[../Notebook/2007-6-12#Mini Prep|Mini Prep]] |
| 3. Resuspend pelleted bacterial cells in 0.3 ml Buffer P1 (in Falcone fridge) and transfer to a microcentrifuge tube
| | *[[../Notebook/2007-6-18#Mini-prep|Mini Prep v.2.0]] |
| * ensure that RNase A has been added to Buffer P1
| | *[[../Notebook/2007-6-12#PCR_amplification_of_EGFP|PCR Amplification]] |
| 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.
| | *[[../Notebook/2007-6-12#PCR_purify(microcentrifuge)|PCR Purification in a Microcentrifuge]] |
| *do not vortex
| | *[[../Notebook/2007-6-12#PCR Cleanup|PCR Cleanup]] |
| * close Buffer P2 immediately after use
| | *[[../Notebook/2007-6-12#Notes for Double Digestion|Double Digestion]] |
| 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.
| | *[[../Notebook/2007-6-13#Ligation|Ligation]] |
| 5. Centrifuge for 10min. at 13,000 rpm
| | *[[../Notebook/2007-6-13#Purifying_the_DNA|Purifying the DNA]] |
| 6. Apply the supernatants from step 4 into the QIAprep spin column by decanding or pipetting.
| | *[[../Notebook/2007-6-14#Transformation_Protocol|Transformation]] |
| 7. Centrifuge for 30-60 seconds on max speed. Discard flow-through
| | *[[../Notebook/2007-7-19#Colony PCR Protocol (OWW, Knight)|Colony PCR (OWW, Knight)]] |
| 8. Wash QIAprep spin column by adding 0.5 ml Buffer PB and centrifuging for 30-60 s. Discard the flow-through.
| | *[[Phosphatase_treatment_of_linearized_vector|Phosphatase treatment of linearized vector]] |
| 9. Discard the flow-through, and centrifuge for an additional 1 min to remove residual wash buffer.
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| 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.
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| === Calculating Optical Density ===
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| Nanodrop Machinery
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| #Start ND-1000 program
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| #Select "nucleic acid"
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| #2 µL water
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| #Push OK
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| #Wipe top and bottom | |
| #2 µL EB buffer (or water if used for elution)
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| #Select "blank"
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| #Wipe top and bottom
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| #2 µl sample
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| #Select "measure"
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| #Record the content of DNA in the lower right hand corner in ng/µl
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| | |
| === PCR ===
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| # Assemble 2 reaction tubes; one with a complete reaction and another without template that serves as a control
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| # Set Reaction Tubes/Plates on Ice
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| # Add the following components in a reaction vessel. Total volume should be between .5-20 µl.
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| #* 45 µl Platinum PCR SuperMix | |
| #* Primers (200 nM final concentration per primer is recommended)
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| #* Template DNA solution
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| # Cap reaction vessel and load into a thermal cycler at 94°C
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| # Incubate tubes in a thermal cycler at 94°C for 30 s to 2 min
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| # Perform the following PCR amplification
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| ## 94°C for 4 minutes
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| ## 94°C for 1 minute
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| ## 55°C for 1 minute
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| ## 72°C for 1 minute
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| ## repeat steps 2-4 35 times
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| ## 72°C for 10 minutes | |
| ## 4°C forever
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| === Purification ===
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| ===General Lab Do s and Don’t s===
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| *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)
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| **Dispensing – go down both notches
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| **Sucking up – go down one notch
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| **Resuspension – mixing by continually sucking up and down (NO BUBBLES)
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| **Look at different numbers on pipettes
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| **If desired, practice on cup of water!
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| *KNOW HOW MUCH ONE MICROLITER IS (for PCR)
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| *Put all enzymes inside cooling blocks next to freezers to avoid denaturation [such as Amp for LB]
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| *Stockroom is also used for carcinogens – DNA staining chemical – don’t touch anything!
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| *Before work: spray ethanol on bench and gloves; Kimwipe (not on open flame!)
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| *Be as sterile as possible! LB is made in building 68 – can pick up more
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| *Automatic Bunsen burners – continuous or sensor
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| *Autoclaved water is sterile
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| ===Growing up bacterial colonies from plates===
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| #Must pick up colonies to grow – inoculation in test tubes – grow over night in media
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| #Put antibiotic in media – all non-plasmid containing bacteria will die (selective amplification)
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| #Antibiotic into LB: 500 LB needs 500 microliters of Amp
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| #Use Bunsen burner, gloves
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| #500 microliters of Amp into LB flask after warming near lip of LB bottle
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| #Discard tips into small buckets on top of bench; dump into red sharps container at end of day
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| #Mark remaining Amp volume with marker
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| #Freezer for iGEM: white, shared for now; later will have freestanding under bench
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| #Use lab tape to re-label and update flasks/containers with substance name, iGEM, date and your initials
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| #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
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| #Transferring LB to blue screw cap – lid of LB down on bench; put LB bottle over Bunsen burner
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| #Temporary fridge space in front of bench
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| #Bacteria with two different types of vectors on streaked plates – make far enough apart to form colony
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| #Make sure automatic pipetter is charging; don’t suck up into device
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| #70% ethanol mix: to 400 with ethanol; rest with distilled water
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| #Glass test tubes of two plasmids – label with name of plasmid and all other relevant details – both cap and tube
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| #Use 10 mL pipette (glass) and automatic pipette (upper button is up) – 5 mL of LB Amp into each tube (inoculation of 5 mL)
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| #Don’t open glass tube cap until necessary
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| #Glass pipettes into liquid-containing plastic waste jar
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| #Flame the metal loop | |
| #Use two fingers to lift cap of glass tube
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| #Flame top of tube before and after touching bacterial colony
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| #Let metal loop cool to temperature of gel by stabbing loop on agar with no colonies
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| #Touch metal loop lightly to one colony and touch to the top of LB broth; mix in broth
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| #A tip: Distinguish before/after inoculation tubes by placing in different rows of the test tube holder
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| #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!
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| #Note the time that incubation starts (for today, 16.29)
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| #If something is growing in culture that should not be there, dump it out after pouring bleach inside
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| #Clean up: Parafilm all containers
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| #LB and Amp go in fridge – must warm up before using next time
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| ===Making stock solution of 1% Agarose in TAE for Gel electrophoresis===
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| #1% agarose gel
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| #Stock agarose solution (for gels)
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| #1 Liter flask
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| #Anything with foil on it is sterile – LB flask does not have to be sterile, since it will be heated anyway
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| #Label 1% agarose, iGEM, TAE buffer, date
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| #1% is weight per volume
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| ##1 mg/mL = 100% [water]
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| ##10 mg/mL = 1%
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| #Get agarose and weigh out 10 mg using boat or paper
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| #Pour agarose into large flask
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| #Clean up weighing area (brush)
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| #Get 1 L graduated cylinder (not sterile) and fill with 1x TAE buffer
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| #Pour in half the TAE buffer from the cylinder into the flask and put in microwave
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| #Wait until bubbling and mix/stir – continue pouring in more buffer until clear
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| #Store in 55ºC box with cap halfway on (no explosions)
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