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| - | <script>
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| - | 1. colony picks.
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| - | 1.label two microcentrifuge tubes one with “E.Coli+PVCFPC-4+LB+GENT” and the other with “E.Coli +PXYFPC-2 +LB+KAN ” and make sure to put the date on each tube.
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| - | 2. pour 30ml LB into tube labeled “E.Coli+PVCFPC-4+LB+GENT” add Gentamicin to final concentration of 15ug/ml.
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| - | 3. pour 30ml LB into tube labeled “E.Coli+PXYFPC-2+LB+KAN” add Kanamycin to final concentration of 30ug/ml.
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| - | 4.take p10 tip , scrape off ONE COLONY off of the plate that reads “LS4260 PXYFPC-2”(in fridge box that says iGEM on it), eject tip into into its respective microcentrifuge tube tube.
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| - | 5. repeat with plate that reads “ LS4342 PVCFPC-4”
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| - | 6. grow overnight at 37C.
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| - | 2. make electrocompetent CB15N?
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| - | 0. grow colony pick overnight CB15N in PYE @ 37C
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| - | 1.7? check OD600 of culture
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| - | 2?balence culture tubes < 0.01 g difference
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| - | 3? spin at 3000 rpm 4C for 15 minutes
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| - | 4? discard supernatant
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| - | 5?add 10 ml Milli Q water
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| - | 6? spin at 3000 rpm 4C for 15 minutes
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| - | 7?discard supernatant
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| - | 8?repeat step 6 and 7 3 times
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| - | 9.? make aliquots and put in deep freezer.
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| - | 3.miniprep on ecoli cultures
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| - | 1.spin ecoli culture at 3000rpm for 5 minutes
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| - | 2.discard supernatant.
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| - | 3. resuspend in 6ml of Mili Q water.
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| - | 4.add 1ml 6x Lysis buffer, invert 4-6 times, let sit for 5 minutes
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| - | 5.add entire mix to blue zymo-midi-filter and column, place in 50ml conical tube. spin 5000 rcf for 6 minutes
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| - | 6.remove and discard filter and place column on collection tube. Place in benchtop centrifuge at max speed for 30 seconds.
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| - | 7.add 400ul endo-wash and spin at max speed on benchtop centrifuge
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| - | 8.add 400ul zippy wash buffer , centifuge at max speed for 1 minute, repeat once.
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| - | 9. transfer to 1.5 ml epedorf tube add 150ul water, let sit for 1 minute before spinning at max speed for 1 minute
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| - | 10. check concentration with nanodrop
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| - | 3.transfect CB15N with plasmids to make two strains of transformed CB15N.
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| - | 4.grow strains on plates.
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| - | 5.pick colonies for overnights.
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| - | 6.induce fluorescence.
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| - | 7.check for fluorescence.
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| - | Gentamycin Dilution
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| - | 1.weigh out .1g Genamycin powder
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| - | 2.place in 50ml microcentrifuge tube
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| - | 3.add 10 ml milli q water
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| - | 4.pull plunger out of 60ml BD syringe
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| - | 5.screw on filter tip.
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| - | 6.fill syringe with solution
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| - | 7.empty syringe into new 50ml microcentifuge tube.
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| - | Sterilized colony pick
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| - | 0. autoclave growth media in container. (allow liberal space in container for aeration of culture), after cooling, add appropriate amount of antibiotic.
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| - | 1.heat tungsten loop on bunsen burner until red hot.
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| - | 2.let cool beside flame
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| - | 3.dip loop in agarose to check temperature
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| - | 4. beneath flame, carefully scrape-off one colony from plate making sure not to get any other colonies on loop.
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| - | 5.beneath flame, stick loop with colony in growth media making sure not to touch the sides of the container with loop. swirl around until colony washes off of loop.
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| - | 6.incubate in appropriate conditions.
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| - | making LB aggar plates (one liter)
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| - | 1. weight out 150 grams of aggar and add water for LB of 150 mg/ml
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| - | 2.autoclave
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| - | 3.cool to ~50C
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| - | 5.add antibiotic
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| - | 6.pour on plates
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| - | 7.leave lid of plates slightly off to reduce condensation and contamination.
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| - | 8.once plates have solidified, stack them with gel facing up
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| - | 9. place in plastic bag to reduce drying out.
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| - | plasmid miniprep
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| - | materials
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| - | -zippy plasmid miniprep kit
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| - | -50 ml conical tubes
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| - | -1.5ml eppendorf tubes
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| - | -mili Q water
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| - | 1. spin down cultures from 9-5-13
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| - | 2. discard supernatant
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| - | 3.add 6ml of mili q water
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| - | 4. resuspend pellets by vortexing
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| - | 5.add 1.5 ml 7x lysis buffer, invert tube 4-6 tubes, sit for 5 minutes.
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| - | 6.add 3.5 ml of cold Neutralization Buffer (yellow), invert 4-6 times after yellow precipitate starts to form, invert 2-3 times.
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| - | 7. place zymo-midi filter/zymo-spin-V-E column assembly into a clean 50 ml conical tube.
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| - | 8.add 400ul of endo-wash buffer and centrifuge at 11,000 for 1 minute . Repeat for 1 minute to eliminate residue ( just column without filter)
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| - | 9.transfer column into 1.5 ml microcentifuge tube and then add 150ul zippy elution buffer centrifuge for 1 minute after incubation for 1 minute.
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| - | Week 1
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| - | 8-1-13
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| - | Made and autoclaved PYE
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| - | prepared another batch of electrocompetent caulobacter (culture OD600 = 0.615A)
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| - | induced KAN resistant with xylose transformed caulobacter @ OD600 = 0.664A
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| - | non induced : 3.25ml PXYFPC-2 + CB15N, 20.5ul antibiotic, 200ml PYE
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| - | induced: 3.25ml PXYFPC-2+ CB15N, 20.5 ul antibiotic, 200ml PYE, .4g xylose .
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| - | electroporation of CB15N with PVCFPC-4 .
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| - | plated on PYE+agarose+gent plates 5 plates (10ul drop, 50ul drop, 100ul drop, two negative contols (see 8-5-13))
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| - | plated CB15N on KAN plate for negative control. (note : this was not prepared following the electroporation protocol with water instead of plasmid. This was just a drop from a colony pick shaking incubator pick of CB15N )
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| - | 8-2-13
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| - | checked fluorescence of PCXFPC-2 : UI (OD600= .76 flouereced, I OD600 = .72 no flourecece.)
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| - | Week 2
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| - | 8-5-13
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| - | checked plates
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| - | neg control (CB15N+KAN electroporated with water): one colony
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| - | neg control (CB15N+KAN straight from culture): many colonies
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| - | 10ul, 50ul, and 100ul many colonies
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| - | plates deemed unusable
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| - | Marshall Porter 8-5-13
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| - | Made new PYE + Gentamycin plates
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| - | 8-6-13
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| - | PXYFPC-2 +CB15N culture to be induced made
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| - | -5ml PXYFPC-2 + CB15N (OD600 = 0.72)
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| - | -20ul KAN
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| - | 50 ul PCR of stpx and pflI on CB15N
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| - | gel electropheresis on PCR reactions
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| - | 8-7-13
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| - | transformation of CB15N with PCXFPC-2 (electroporation) time constant = 5.6
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| - | transformation of CB15N with PVCFPC-4 (electroporation) time constant = 5.4
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| - | PXYFPC-2 +CB15N culture to be induced made
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| - | -5ml PXYFPC-2 + CB15N (OD600 = 0.72)
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| - | -20ul KAN
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| - | Marshall Porter 8-7-13
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| - | ● TD PCR for Stpx and pflI tags from caulobacter, two 15 cycle sets
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| - | ● initial denature of 95 C
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| - | denature anneal elongation denature anneal elongation
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| - | 95 C 56 C 68 C 95 C 49 C 68 C
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| - | :10 :15 2:20 :10 :15 2:20
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| - | Gel?...unsucessful
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| - | Week 3
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| - | 8-13-13
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| - | TD PCR for Stpx and pflI #3, 4, and 5. Two sets of 15 cycles with initial denature at 95 C for 1:00 for #3 and 94 C for 3:00 for #4 and #5
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| - | #3
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| - | denature anneal elongation denature anneal elongation
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| - | 95 C 56 C 68 C 95 C 49 C 68 C
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| - | :10 :15 2:20 :15 :15 2:20
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| - | #4
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| - | denature anneal elongation denature anneal elongation
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| - | 94 C 56 C 68 C 94 C 49 C 68 C
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| - | :15 :30 2:20 :15 :30 2:20
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| - | #5
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| - | denature anneal elongation denature anneal elongation
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| - | 94 C 59 C 68 C 94 C 52 C 68 C
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| - | :15 :15 2:30 :15 :15 2:30
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| - | 8-15-13
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| - | made LB
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| - | checked plates?
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| - | 8-19-13
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| - | Gentamicin dilution (10mg/ml)
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| - | 1.weigh out .1g genamycin powder
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| - | 2.place in 50ml microcentrifuge tube
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| - | 3.add 10 ml mili q water
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| - | 4.pull plunger out of 60ml BD syringe
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| - | 5.screw on filter tip
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| - | 6.fill syringe with solution
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| - | 7.empty syringe into new 50ml microcentrifuge tube.
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| - | 8.aliquot 100ul, 10ul, 500ul, and 10ul into 1.5ml ependorf tubes
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| - | sterilized enoculation of LB with ecoli + PXYFPC-2 +KAN (30ug/ml final concentration)
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| - | sterilized enocultion of LB with ecoli + PVCFPC-4 +GENT (15ug/ml final concentration)
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| - | 8-20-13
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| - | Glycerol stock of E. coli cultures containing PVCFPC-4
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| - | Plasmid Midi-prep of E. coli cultures
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| - | ● Culture #1 OD550 .916
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| - | ● Culture #2 OD5050 2.149
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| - | Nucleic Acid Concentration A260 A280 260/280 260/230
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| - | Culture #1 21.0 ng/ul .419 .221 1.89 1.68
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| - | Culture #2 299.2 ng/ul 5.983 3.184 1.88 2.13
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| - | Week 5
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| - | 9-?-13
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| - | use genious to design primers (forward and reverse) to get Halorhodopsin (HsHR) out of H.Salinuarium.
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| - | order primers.
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| - | 9-3-13
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| - | take chip of frozen glycerol stock H.Salinarium and thaw in ?
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| - | 9-4-13
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| - | 25 ul PCR using HsHR primers ,HS (template), and GC master mix.
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| - | negative control without primers labeled (-1)
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| - | negative control without HS (template) labeled (-2)
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| - | 98C -2mins/98C 10s 57.2C 72C 2 min/72C 3min hold 4C
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| - | results gel?
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| - | 9-5-13
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| - | sterilized inoculation of SOC with iGEM part BBa_K9000 “9000” +ecoli
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| - | sterilized enoculation of SOC with iGEM part BBa_K9001 “9001” +ecoli
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| - | sterilized enoculation of SOC with iGEM part BBa_K9010 “9010” +ecoli
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| - | made chloramphenicol (1ul/1ml) plates
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| - | PCR of Halorhodopsin from Halobacterium Salinarum 30 cycles
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| - | 25 ul reaction with 2x phusion GC Master Mix
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| - | initial denature denature anneal extension final extension
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| - | 98 C 98 C 57 C 72 C 72 C
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| - | 2:00 00:10 00:20 1:00 5:00
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| - | 9-6-13
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| - | plasmid miniprep on 9000, 9010, and 9010 cultures.
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| - | results
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| - | -9000 = 99.2 ng/ml
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| - | -9001 =99.7 ng/ml
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| - | -9010 = 38.1 ng/ml
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| - | gel of parts
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| - | gell legend : 1Kb ladder , 9010, 9001, 9000
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| - | design and order sequencing primers for iGEM parts. same forward and reverse for all three.
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| - | Week 6
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| - | 9-9-13
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| - | send plasmids with sequencing primers out for sequencing.
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| - | design and order primers for plasmid backbone.
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| - | 20ul PCR on HS for HsHR
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| - | run diagnostic gel
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| - | -gel legend : 1Kb ladder , H? , +, -1, -2,
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| - | 9-10-13
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| - | 50 ul HsHR PCR labeled “50”
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| - | 98C 2min/ 98C :10s/57.2C 72 1min/72min/Hold 4C
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| - | 9-11-13
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| - | purification gel
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| - | -gel legend: 1Kb ladder, 50
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| - | 50ul HsHR PCR (two reactions “50(1)” and “50(2)”) one negative control with no template “50 -”
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| - | gel legend : 1Kb ladder , 50(1), 50(2), 50-
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| - | 9-12-13
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| - | 50ul PCR to obtain plasmid backbone using 9010 (30.4ng/ul) as template.
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| - | initial denature denature anneal extension final extension
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| - | 98 C 98 C 52.2 C 72 C 72 C
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| - | :30 :10 :30 1:00 5:00
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| - | gel legend : 1Kb ladder , 50(1), 50(2), 50-
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| - | Two 20ul PCR reactions using part 9010 with different annealing temperatures. One at 55 C and one at 57 C
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| - | gel legend : 1Kb ladder , 9010(55 C) , 9010 neg (55 C), 9010 (57 C), 9010 neg (57C)
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| - | 9-14-13
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| - | 20ul PCR to obtain plasmid backbone using linearized backbone as template.
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| - | gel legend : 1Kb ladder , 9010 57 C , 9010 57 C(-), linear 57, linear 57 neg
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| - | Week 7
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| - | 9-15-13
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| - | New PCR of 9010 at 55 C and 57 C with new GC phusion mastermix due to contamination
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| - | and a PCR of Linear PSB1C3 at 57 C
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| - | gel legend : 9010(55+), 9010(55 C-) , 9010(57 C+), 9010(57 C-), Linear (1), Linear (2), Linear(-), DNA ladder
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| - | 9-16-13
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| - | Gel purification on PCR with linearized backbone
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| - | ● Run two samples from the PCR reaction, one with 5ul pcr and loading buffer with sybr gold, and the other with the rest of the pcr (45ul) and loading buffer without sybr gold.
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| - | ● Load the two samples in side by side lanes
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| - | ● Use the sybr gold in uv light to find the band to cut out of the gel in the next lane over.
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| - | ● Centrifuge gel cut out with gel filter to separate. Collect filtrate.
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| - | ● Use Zymo research DNA Clean and Concentrate to recover DNA from filtrate
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| - | 9-17-13
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| - | concentrate DNA
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| - | 1.combine all gel purification tubes with purified DNA (add alcohol?)
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| - | 2.place sepreate cap with holes in it on top of tube.
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| - | 3.place tube in brick under vacuum chamber.
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| - | 4.when all liquid is gone, add 5ul of Milli Q water.
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| - | 9-18-13
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| - | gibson reaction with KR2 G-blocks
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| - | -5ul concentrated plasmid backbone
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| - | -1ul of each G-block
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| - | -3ul H2O
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| - | -10ul 2x Gibson Master Mix
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| - | -PCR Machine 50C for 1 hr.
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| - | chemicompetent transformation of e.coli with Gibson reaction products.
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| - | plate transformed e.coli on LB+Chloramphenicol plates. (400ul , 200ul, 100ul, and one with untransformed ecoli)
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| - | 9-19-13
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| - | suspend colonies in mili q water
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| - | mili q water with colonies colony PCR on colonies from 400ul and 200ul plates
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| - | 15 cycles
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| - | 94 C initial denature 5 minutes
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| - | 94 C denature 15 s
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| - | 55 C first anneal 30s
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| - | 68 C extension 1:00
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| - | 15 cycles anneal drop down 7 C
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| - | 94 C denature :15
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| - | 48 C second anneal :30
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| - | 68 C extension 1:00
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| - | 68 C final extension 5:00
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| - | 4 C hold infinite
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| - | inoculation of e. coli colonies in LB + chloramphenicol
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| - | 9-20-13
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| - | run gel on PCR products from 9-19-13
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| - | gel legend: 1Kb ladder, “400”, “200”, negative control (no template)
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| - | Zymo research plasmid midi-prep from e. coli cultures
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| - | 9-21-13
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| - | Send psb1c3+KR2 plasmid in for sequencing.
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| - | 9-24-13
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| - | receive sequencing results (not sure how to explain the results)
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| - | <script>
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| | Reference Sequences | | Reference Sequences |
LB Media Recipe
Making LB+Kanamycin and LB+Gentamicin Agar Plates: To make a 500 ml batch: - 475 ml of MilliQ water - 5 g of Tryptone - 5 g of NaCl - 2.5 g of yeast Extract - 7.5 g of Agar We made x2 500 ml batches. Combine the reagents and shake until the solutes have dissolved. Adjust the ph to 7.0. Sterilize by autoclaving for 20 minutes on liquid cycle. Take out of the autoclave and let the solution cool. Place in a water bath to prevent solid formation. Add 1.25 ml of gentamicin antibiotic to one of the 500 ml batches and 500 ul of kanamycin to the other batch. Pour into petri dishes about half way. Let agar solidify. Place in the refrigerator.
Making Top10 Chemicompetent Cells:
1) Grow Top10 SOB overnight between the temperatures 20°C and 33°C.
2) Measure the OD and bring the OD back to 0.1.
3) Add 2.5 ml of Top10 SOB to SOB.
4) Incubate at 31°C for one hour and 20 minutes or until the OD is 0.6.
5) Create 5 ml of 1X Washing buffer: 2.5 ml of Dilution buffer and 2.5 ml of Wash buffer (2X)
6) Create 5 ml of 1X Competent buffer: 2.5 ml Dilution buffer and 2.5 ml of Competent buffer (2X)
7) Incubate the culture on ice for ten minutes and then pellet cells at 2,500 xg for ten minutes at 0-4°C.
8) Remove supernatant. Gently resuspend cells in 5 ml of ice cold 1X Wash buffer. Re-pellet as in step 7.
9) Remove supernatant completely. Gently resuspend cells in 5 ml of ice cold 1X Competent buffer.
10) Aliquot 100-200 ul. Flash freeze.
11) Store in -80°C freezer.
Making LB+Kanamycin and LB+Gentamicin Agar Plates:
To make a 500 ml batch:
- 475 ml of MilliQ water
- 5 g of Tryptone
- 5 g of NaCl
- 2.5 g of yeast Extract
- 7.5 g of Agar
We made x2 500 ml batches.
Combine the reagents and shake until the solutes have dissolved. Adjust the ph to 7.0. Sterilize by autoclaving for 20 minutes on liquid cycle.
Take out of the autoclave and let the solution cool. Place in a water bath to prevent solid formation.
Add 1.25 ml of gentamicin antibiotic to one of the 500 ml batches and 500 ul of kanamycin to the other batch.
Pour into petri dishes about half way. Let agar solidify. Place in the refrigerator.
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