IGEM:MIT/2006/Index

Projects/Progress

1. Preliminary Research
   • Determine toxicity of precursors and products (VV) (complete)
     • [[../Notebook/2006-6-16#Toxicity |6-16-2006]] covered toxicity
   • Test effects of minimal media on smell (complete)
     • Reduced smell with no tryptophan, [[../Notebook/2006-6-20#WINTERGREEN!!!!!!! |6-20-2006]] - now testing other mediums to find best
     • Determine optimal concentration of SA: 400 uL
   • Determine the origin of the bad smell (indole?) (complete)
     • Indole is bad smell, [[../Notebook/2006-6-20#Indole |6-20-2006]] - now looking to get knockouts
   • Explore biosynthesis of precursors
   • Sequence BAMT, SAMT, BSMT
     • From initial Top10 with [[../Notebook/2006-6-13#pET-28 Primers |T7/TPhiR]] on [[../Notebook/2006-6-16#Sequencing |6-16-2006]]
     • Troubleshoot SAMT with [[../Notebook/2006-6-8#SAMT from A.Majus |designed reverse primer]] on [[../Notebook/2006-6-16#Sequencing |6-16-2006]]
2. Expression in E. coli
   • Transformation into Top10 (complete)
   • Transformation into BL21 (DE3)
     • [[../Notebook/2006-6-16#BL21 Tranformation |6-16-2006]]
   • Indole knockout
3. Applications
   • Heat promoter
4. Yeast incorporation
   • Discussion at [[../Notebook/2006-6-19#Yeast_Incorporation |6-19-2006]]
   • Preliminary experiments with ATF1 [[../Notebook/2006-6-15 |6-15-2006]] and [[../Notebook/2006-6-19#Repeat ATF1 PCR |6-19-2006]]
5. Quantification
   • Get trained for GC/MS use
     • Discussion at [[../Notebook/2006-6-14#Resources_on_gas_chromatography-olfactometry |6-14-06]]
     • Documentation on the appropriate instrumentation and procedure for GC with methyl benzoate here

Protocols

1. PCR - Veena
   • First, you might want to dilute some of your miniprep product (that is, your plasmid DNA...this is your template) to a concentration of 1ng/μL, and label this tube; for example, if I have BSMT plasmid DNA that I got from a miniprep and it's at a concentration of 50ng/μL, I'd label a new tube "BSMT for PCR" and add to that tube 49μL of water and 1μL of the 50ng/μL plasmid DNA.
   • You should also prepare 25μM tubes of primer, if you don't have them already.
   • In each tube, you want a total reaction volume of 50μL, with 1ng of your template DNA, .6μL of each primer (forward and reverse) (.6 μL of the 25μM primer yields roughly 300 nM final concentration of that primer, which is the goal), and 49 μL of Tom's PCR mix.
   • Run the tubes on the thermocycler at: 95deg for 3:00, then cycle through: (a) 94deg for :30 (b) 55deg for :30 (c)68deg for 2:15, then 72deg for 10:00
2. Digestion - Kate [[../Notebook/2006-6-27#Restriction Digest |6-27-2006]]
   • Important Notes:
     • Keep all enzymes and buffers on ice for the entire process
     • Want in each tube a total volume of 50 μL
     • if cutting for a biobrick fabrication, use EcoR1 and Pst1 restriction enzymes (.5 -1 μL of enzyme is okay to use, just remember that too much glycerol in your digestion reaction could potentially cause unwanted star activity)
     • use Dpn1 only if you are cutting a PCR product
     • Digest your PCR insert and backbone separately, and also digest a control part in parallel to check that digest is working correctly
   • Reaction Procedure steps: (50 μLL scale) --- in order of addition
     • ?? μL Water (to make 50 μL)
     • 5 μL Buffer 2 (quick vortex first)
     • .5 μL BSA (quick vortex first)
     • ?? μL DNA (whatever neccesary to get 800 ng -- good idea to do a fresh nanodrop of your sample and then make this calculation)
     • .5 μL Pst1 and .5 μL EcoR1
     • .5 μL Dpn 1 (only if digesting a PCR product)
   • Post reaction steps:
     • incubate at 37c for 2 hours (1 minimum and 6 maximum)
     • heat shock for 20 mins at 80c to heat inactivate the enzyme, then store at 4c forever
     • do a purification (elute in 30μL) just like a pcr clean up
     • run a gel to check this step before moving on to ligation reaction
3. Ligation - Veena [[../Notebook/2006-6-21#Ligation reaction |6-21-2006]] Knight
4. Transformation - André Transform competent cells
5. Sequencing - Bo
   • Reconstitute primer of X nMoles by adding 10*X uL of water, making 100 uM solution (can turn into 25 uM by using 40*X)
   • Centrifuge for 15 seconds.
   • Add 180ng of template DNA
   • Add 3.2 pMoles of primer (0.128 uL from 25 uM working solution)
6. Pouring Plates - Kate
   • Preparation of LB Agar
     • Obtain 1.2% LB agar from the media room (500 ml per bottle).
       • Be sure to sign it out
     • Melt in microwave:
       • loosen the cap a lot, lay down paper towels in case of spill, use 50% power (enter time, press Power, 5, Start)
       • monitor as you melt, takes approx. 10 minutes per bottle (if doing 2 or 3 bottles simultaneously, add 5 minutes)
     • If too hot, let the melted solution cool so that it's warm, but not hot to the touch - you should use the water bath to do this (50-60 degrees); this won't let it resolidify.
   • Once it's cool enough (50-60 degrees) - then add antibiotics: final concentration, 50ug/ml AMP; 20ug/ml KAN. (concentrations of liquid stocks: Amp; 50mg/ml. Kan, 10mg/ml.)
     • For 500mL of LB Agar use:
       • 500μL Amp stock
       • 1mL Kan stock
     • Swirl to mix; try not to make many bubbles.
   • Actual Pouring
     • Obtain a container of empty plates. One bottle (500ml) of LB Agar will make about one container of plates (20 plates).
     • Cover the base of the plate, and then just a bit more after that.
     • Recap each plate upon pouring. If there are lots of bubbles in your plates (i.e., more than one or two on the edge), you can flame the plate using the small bunsen burner to eliminate bubbles. Another way to remove the fine bubbles that may be in your flask before puring is to mist the inside of the flask with a 75% ethanol spray bottle.
     • Leave plates to dry and cool for a while on the benchtop (overnight even).
       • It is a good idea to label the stack of plates to indicate antibiotic.
     • Store the plates in their original bags - upside down, so that the gel is hanging downwards (this keeps condensation off the gel).
       • Label the bags following taping rules:
         • Red tape in back (indicates LB)
         • Tape in front indicates antibiotic (green=Kan, yellow=Amp, more taping/color rules are on the refrigerator in 68-564)
         • Also write name of antibiotic, and concentration, on the front piece of tape.
       • Store the labeled bags of plates in the fridge
7. Autoclaving (waste, media, etc.) - Stephen
8. Pouring/running gels - Veena
   • First, you have to pour the gel:
     • Get an empty small flask, and the 1% agarose from the 70-something degree incubator by the door (watch out...the agarose is hot. You might want to wear gloves)
     • Flame the bottom of the flask to heat it up (for maybe 5-10 seconds? Not too long! the point of this is just to make sure that the gel doesn't solidify in the beaker before you pour it)
     • Pour about 40mL of the 1% agarose into the flask; put the agarose back in the incubator
     • put on gloves and take the flask containing the 40mL of agarose to the gel room
     • carefully add 2μL of Ethidium Bromide to the agarose, making sure to keep the vial of EB in its plastic container; swirl the flask gently to mix the EB
     • press a small plastic gel holder into the long plastic u-shaped thing (hard to describe...you'd probably have to see this demonstrated), and place a purple plastic gel-well-making thing in the top slot (put it in with the side with the fat purple things pointing downward)
     • pour the agarose + EB from the flask carefully into the gel-holder. Try to avoid making bubbles, and make sure the agarose isn't gloppy.
     • General notes: you want to try to work quickly, since you don't want the gel to solidify before you pour it
9. Glycerol stocks - Stephen