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

Digestion - Kate [[../Notebook/2006-6-27#Restriction Digest |6-27-2006]]

1. • 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 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: (50 μLL scale) --- in order of addition
     • Water (?? μL to make 50 μL)
     • Buffer 2, 5 μL (quick vortex first)
     • BSA, .5 μL (quick vortex first)
     • DNA, ?? μL (whatever neccesary to get 800 ng -- good idea to do a fresh nanodrop of your sample and then make this calculation)

incubate at 37c for about 2 hours (1 minimum and 6 maximum) do a purification (elute in 30μL) just like a pcr clean up

lkdfj

1. Ligation - Veena [[../Notebook/2006-6-21#Ligation reaction |6-21-2006]] Knight
2. Transformation - André Transform competent cells
3. 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)
4. Pouring Plates - Kate
5. Autoclaving (waste, media, etc.) - Stephen
6. Pouring/running gels - Veena
7. Glycerol stocks - Stephen