IGEM:MIT/2007/Protocols: Difference between revisions

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=== Calculating Optical Density ===
==General Lab Protocols==
Nanodrop Machinery
*[[../Notebook/2007-6-11#General_Lab_Do_s_and_Don’t_s|General Lab Do's and Don'ts]]
#Start ND1000 pprogram
*[[../Notebook/2007-6-12#Nanodrop (~20ng/ul) Protocol|Calculating Optical Density; Nanodrop Protocol]]
#Select "nucleic acid"
*[[../Notebook/2007-6-12#Obtaining a Cast|Making a Cast]]
#2uL water
*[[../Notebook/2007-6-11#made_1_liter_1%_agarose_solution|Making Agarose Solution]]
#Push OK
*[[../Notebook/2007-6-12#Protocol for Preparing Agarose Gel|Making an Agarose Gel]]
#Wipe top and bottom
*[[../Notebook/2007-6-12#Protocol for Preparing to Run a Gel Electrophoresis|Preparing a Gel Electrophoresis]]
#2uL EB buffer (or water if used for elution)
*[[../Notebook/2007-6-12#Protocol for Running a Gel Electrophoresis|Running a Gel Electrophoresis]]
#Select "blank"
*[[../Notebook/2007-6-13#Making_LB_Amp_Agar_Plates|Making LB Amp Agar Plates]]
#Wipe top and bottom
*[[../Notebook/2007-6-11#innoculate_5ml_LB/Amp_with_bact._colonies_(EGFP_and_NNX)|Innoculating LB with bacteria]]
#2ul sample
*[[../Notebook/2007-6-12#Protocol for Imaging Result and Gel Diagnostic|Gel Imaging]]
#Select "measure"


=== PCR ===
==DNA Work==
## Assemble 2 reaction tubes; one with a complete reaction and another without template that serves as a control
*[[../Notebook/2007-6-12#Mini Prep|Mini Prep]]
## Set Reaction Tubes/Plates on Ice
*[[../Notebook/2007-6-18#Mini-prep|Mini Prep v.2.0]]
## Add the following components in a reaction vessel. Total volume should be between .5-20 µl.
*[[../Notebook/2007-6-12#PCR_amplification_of_EGFP|PCR Amplification]]
### 45 µl Platinum PCR SuperMix
*[[../Notebook/2007-6-12#PCR_purify(microcentrifuge)|PCR Purification in a Microcentrifuge]]
### Primers (200 nM final concentration per primer is recommended)
*[[../Notebook/2007-6-12#PCR Cleanup|PCR Cleanup]]
### Template DNA solution
*[[../Notebook/2007-6-12#Notes for Double Digestion|Double Digestion]]
## Cap reaction vessel and load into a thermal cycler at 94°C
*[[../Notebook/2007-6-13#Ligation|Ligation]]
## Incubate tubes in a thermal cycler at 94°C for 30 s to 2 min
*[[../Notebook/2007-6-13#Purifying_the_DNA|Purifying the DNA]]
## Perform the following PCR amplification
*[[../Notebook/2007-6-14#Transformation_Protocol|Transformation]]
### 94°C for 4 minutes
*[[../Notebook/2007-7-19#Colony PCR Protocol (OWW, Knight)|Colony PCR (OWW, Knight)]]
### 94°C for 1 minute
*[[Phosphatase_treatment_of_linearized_vector|Phosphatase treatment of linearized vector]]
### 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)

Latest revision as of 08:10, 20 July 2007

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]]

DNA Work

  • [[../Notebook/2007-6-12#Mini Prep|Mini Prep]]
  • [[../Notebook/2007-6-18#Mini-prep|Mini Prep v.2.0]]
  • [[../Notebook/2007-6-12#PCR_amplification_of_EGFP|PCR Amplification]]
  • [[../Notebook/2007-6-12#PCR_purify(microcentrifuge)|PCR Purification in a Microcentrifuge]]
  • [[../Notebook/2007-6-12#PCR Cleanup|PCR Cleanup]]
  • [[../Notebook/2007-6-12#Notes for Double Digestion|Double Digestion]]
  • [[../Notebook/2007-6-13#Ligation|Ligation]]
  • [[../Notebook/2007-6-13#Purifying_the_DNA|Purifying the DNA]]
  • [[../Notebook/2007-6-14#Transformation_Protocol|Transformation]]
  • [[../Notebook/2007-7-19#Colony PCR Protocol (OWW, Knight)|Colony PCR (OWW, Knight)]]
  • Phosphatase treatment of linearized vector
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