Team:Illinois/Antibody Receptor Tyrosine Kinase Fusion Notebook

From 2008.igem.org

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{{bottom_template}}
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mZgAkl  <a href="http://fbdnisdgysij.com/">fbdnisdgysij</a>, [url=http://jzowycxvfekb.com/]jzowycxvfekb[/url], [link=http://lkctxlgmquum.com/]lkctxlgmquum[/link], http://obyjjyzxsbxr.com/
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== [https://2008.igem.org/Illinois/1_July_2008 July 1, 2008] ==
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Made Yeast Media
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YPD Medium, per liter:
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10g yeast extract
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20g peptone
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20g dextrose
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20g agar(only for plates)
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1. Dextrose filter sterilized, the rest autoclaved
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2. Weigh nutrients into flask double the volume you want to make, and stir to dissolve
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3. Dextrose added to autoclaved media to equivalent of 20 g/L
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4. Liquid media placed on bench, plate media placed in 65 degree water bath approximately 5 minutes
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5. Poured into plates and allowed to solidify
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== [https://2008.igem.org/Illinois/17_July_2008 July 17, 2008] ==
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E.Coli Media
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LB medium, per liter
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10g tryptone
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5g yeast extract
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5g NaCl
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1 mL 1N NaOH
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15g (agar for plates)
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1. Antibody DNA resuspended in TE buffer, 0.1 μg/μL
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2. 5mL LB inoculated with single colony DH5a pro
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3. Incubated at 37 degrees overnight
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== [https://2008.igem.org/Illinois/18_July_2008 July 18, 2008] ==
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Competent cells:
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1. 3mL overnight culture of DH5a pro
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2. Inoculated into 35mL of LB
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3. OD600 checked, want 0.2-0.3
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4. Place culture on ice for 3 minutes
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5. Spin at 10,000 rpm for 7 minutes, discard supernatant
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6. Resuspend in 10 mL cold 30 mM CaCl2, incubated on ice overnight
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7. Glycerol added to 10%, cells in freezer
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== [https://2008.igem.org/Illinois/19_July_2008 July 19, 2008] ==
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Transformation:
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1. Cells put in ice 30 min with 1μL plasmid(100ng)
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2. Heat Shock for 2 minutes at 42 degrees
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3. Ice for 8 minutes
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4. Add cells to 1ml LB
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5. Grow for 1 hour
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6. Plate 100 to 200 μL on LB and amp, grow overnight
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== [https://2008.igem.org/Illinois/21_July_2008 July 21, 2008] ==
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Observations:
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Plenty of colonies on all plates
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Also, making 5mL overnight cultures for mini-prep tomorrow.
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== [https://2008.igem.org/Illinois/22_July_2008 July 22, 2008] ==
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Mini spin prep on LC and HC colonies
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Place DNA in 100μL H2O in freezer
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== [https://2008.igem.org/Illinois/29_July_2008 July 29, 2008] ==
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All primers brought to standard concentration, 30mM
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33.3μL H2O added per n mole of primer
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== [https://2008.igem.org/Illinois/30_July_2008 July 30, 2008] ==
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Assembly PCR: amplify antibody gene fragments and then assemble them, light and heavy chains
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{| class="wikitable" border="1"
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|tube
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|1
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|2
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|3
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|4
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|5
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|6
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|7
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|8
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|-
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|Antibody chain
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|H
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|H
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|L
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|L
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|H
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|H
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|L
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|L
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|DNA source
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|colspan="4"|mini-prep
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|colspan="4"|synthesized IDT genes
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|-
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|template
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|colspan="8"|0.5ul
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|primers
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|colspan="8"|1ul of appropriate forward and reverse primer
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|MgCl2
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|3ul
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|5ul
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|3ul
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|5ul
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|3ul
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|5ul
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|3ul
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|5ul
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|master mix
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|colspan="8"|20ul
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|H20
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|colspan="8"|to 50ul total volume
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|}
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Master mix already contains MgCl2; should have added extra to some tubes.
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PCR program
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# 94 degrees 5 min
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# 94 degrees 1 min
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# 50 degrees 1 min
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# 72 degrees 1 min
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# GOTO 2. 29 cycles
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# HOLD at 4 degrees
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1.5% agarose gel made, 0.75g agarose in 50ml 0.5X TBE w/10ul EtBr.
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20ul of PCR produts loaded on gel w/4ul 6X loading buffer, run at 200V for about half an hour.
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All lanes had lots of DNA at ~375bp, PCR worked.
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[[Image:07-30-08_AbFrag_Gel1.jpg|none]]
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== [https://2008.igem.org/Illinois/31_July_2008 July 31, 2008] ==
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PCR to asemble fragments from yesterday.
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{| class="wikitable" border="1"
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|tube
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|1
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|2
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|3
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|4
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|template from 7-30 PCR
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|2+3
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|2+7
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|6+7
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|1+8
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|template
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|colspan="4"|0.5ul of both heavy and light chains
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|primers
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|colspan="8"|1ul of appropriate forward and reverse primer
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|MgCl2
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|0ul
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|0ul
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|3ul
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|3ul
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|master mix
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|colspan="4"|20ul
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|H20
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|colspan="4"|to 50ul total volume
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|}
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PCR program
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# 94 degrees 5 min
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# 94 degrees 1 min
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# 50 degrees 1 min
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# 72 degrees 1 min
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# GOTO 2. 29 cycles
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# 72 degrees 5 min
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# HOLD at 4 degrees
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Products run on a 1.5% agarose gel, no products of 700-800bp.
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[[Image:7_31_08_assembly.jpg|none]]
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== [https://2008.igem.org/Illinois/1_August_2008 August 1, 2008] ==
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PCR troubleshooting of yesterday's reaction:
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use gradient for annealing temp (40-65 degrees), use more template, leave out end primers
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Four series of reactions using a gradient annealing temp were run in addition to a fifth series omitting the end primers.
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{| class="wikitable" border="1"
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|reaction series
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|A
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|B
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|C
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|D
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|E
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|template (5+7) from 7-30 PCR
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|0.5ul
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|1ul
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|1.5ul
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|2ul
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|1ul
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|primers
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|colspan="5"|1ul of forward and reverse primers (omitted in E series)
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|master mix
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|colspan="5"|20ul
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|H20
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|colspan="5"|to 50ul total volume
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annealing temps: 40, 43.8, 50, 54, 60, and 64 degrees
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PCR program
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# 94 degrees 5 min
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# 94 degrees 1 min
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# annealing step 1 min
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# 72 degrees 1 min
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# GOTO 2. 29 cycles
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# HOLD at 4 degrees
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PCR products run on 1.5% agarose gel at 200V for ~20 minutes, then 240V for ~20 minutes.
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Products in the 700-800bp range in all lanes.  Perhaps it worked because of using a differnt product from the 7-30 PCR.
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[[Image:8_1_08_assembly_gradient.jpg|none]]
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== [https://2008.igem.org/Illinois/6_August_2008 August 6, 2008] ==
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1.5% agarose gel of 8-1 PCR products run to cut out bands. Products A1, B1, C1, and E1 used.
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Bands around 800bp cut out for all lanes and invitrogen gel extraction kit used to collect DNA in 50ul H2O.
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== [https://2008.igem.org/Illinois/5_September_2008 September 5, 2008] ==
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Flk-1 clones from [http://www.atcc.org/ATCCAdvancedCatalogSearch/ProductDetails/tabid/452/Default.aspx?ATCCNum=MGC-18600&Template=mgcMouseClones ATCC] streaked onto LB+amp and incubated at 37 degrees.
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== [https://2008.igem.org/Illinois/6_September_2008 September 6, 2008] ==
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Colonies picked from flk-1 plate and inoculated into 5ml LB+amp, grown at 37 degrees.
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Freeze-dried e. coli with YCp50-poly from [http://www.atcc.org/ATCCAdvancedCatalogSearch/ProductDetails/tabid/452/Default.aspx?ATCCNum=87555&Template=vectors ATCC] resuspended in 5ml LB, grown at 37 degrees.
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== [https://2008.igem.org/Illinois/7_September_2008 September 7, 2008] ==
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Mini spin prep of flk-1 and YCp50-poly cells, plasmids stored in 50ul H2O.
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== [https://2008.igem.org/Illinois/25_September_2008 September 25, 2008] ==
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Biobricks
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Bricks to be made: H chain, L chain, SCA, flk-1
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{| class="wikitable" border="1"
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|tube
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|6
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|7
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|8
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|0.5uL template
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|H chain
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|L chain
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|SCA
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|SCA
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|SCA
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|SCA
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|flk-1
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|flk-1
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|IDT
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|IDT
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|A
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|B
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|C
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|E
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|A
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|B
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|primers
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|colspan="8"|1uL forward & reverse for all
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|mastermix
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|colspan="8"|8.25uL for all
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|H2O
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|colspan="8"|39.25uL for all
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{| class="wikitable" border="1"
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|tube
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|9
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|10
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|11
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|12
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|13
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|14
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|0.5uL template
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|SCA w/ link
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|SCA w/ link
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|SCA w/ link
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|SCA w/ link
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|flk link 1
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|flk link 1
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|A
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|B
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|C
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|E
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|A
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|B
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|primers
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|colspan="8"|1uL forward & reverse for all
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|mastermix
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|colspan="8"|8.25uL for all
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|H2O
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|colspan="8"|39.25uL for all
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|}
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{| class="wikitable" border="1"
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|tube
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|15
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|16
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|17
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|18
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|0.5uL template
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|flk link 2
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|flk link 2
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|flk link 3
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|flk link 3
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|-
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|A
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|B
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|A
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|B
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|-
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|primers
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|colspan="8"|1uL forward & reverse for all
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|mastermix
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|colspan="8"|8.25uL for all
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|H2O
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|colspan="8"|39.25uL for all
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|}
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PCR program
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# 94 degrees 4 min
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# 94 degrees 1 min
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# 50 degrees 1 min
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# 72 degrees 3 min
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# GOTO 2. 29 cycles
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# HOLD at 4 degrees
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== [https://2008.igem.org/Illinois/26_September_2008 September 26, 2008] ==
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*product from yesterday run on 1% gel, 200V for ~30 minutes
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*mostly good results, small amound of DNA for flk-1
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*lanes 1,2,6,8,12,14,16,18 cut out and purified with invitrogen gel extraction kit, eluted in 50ul H2O
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<br>
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[[Image:9_26_08_biobricks.jpg|none]]
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<br>
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[[Image:9_26_08_biobricks_(2).jpg|none]]
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PCR to assemble SCA-flk-1 fusion and increase flk-1 yield
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{| class="wikitable" border="1"
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|tube
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|1
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|2
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|3
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|4
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|5
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|6
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|7
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|8
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|9
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|10
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|11
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|12
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|13
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|14
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|15
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|16
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|17
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|18
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|19
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|20
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|template
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|0.5ul flk-1 A
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|0.5ul flk-1 B
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|0.5ul flk link 1 A
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|0.5ul flk link 1 B
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|0.5ul flk link 2 A
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|0.5ul flk link 2 B
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|0.5ul flk link 3 A
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|0.5ul flk link 3 B
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|0.5ul SCA link + 0.5ul flk link 1
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|0.5ul SCA link + 0.5ul flk link 1 (crude)
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|0.5ul SCA link + 2ul flk link 1
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|0.5ul SCA link + 2ul flk link 1 (crude)
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|0.5ul SCA link + 0.5ul flk link 2
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|0.5ul SCA link + 0.5ul flk link 2 (crude)
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|0.5ul SCA link + 2ul flk link 2
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|0.5ul SCA link + 2ul flk link 2 (crude
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|0.5ul SCA link + 0.5ul flk link 3
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|0.5ul SCA link + 0.5ul flk link 3 (crude)
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|0.5ul SCA link + 2ul flk link 3
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|0.5ul SCA link + 2ul flk link 3 (crude)
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|-
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|primers
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|colspan="20"|1uL forward & reverse for all
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|-
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|mastermix
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|colspan="20"|8.25uL for all
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|-
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|H2O
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|colspan="20"|to 50ul total volume
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|}
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PCR program
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# 94 degrees 4 min
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# 94 degrees 1 min
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# 50 degrees 1 min
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# 72 degrees 3 min
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# GOTO 2. 29 cycles
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# HOLD at 4 degrees
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== [https://2008.igem.org/Illinois/27_September_2008 September 27, 2008] ==
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*0.8% agarose gel run of products from yesterday, 200V for ~45 minutes
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<br>
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[[Image:9_27_08.jpg|none]]
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*first 8 reactions may have worked but bands not great, assembly reactions did not work
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<!-- == Insert Date Here ==
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* lab procedure
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** more lab procedure
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* second lab procedure
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** more about second lab procedure
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COPY/PASTE THEN REMOVE THIS AND BRACKETS-->
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{{bottom_template}}
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Latest revision as of 06:22, 29 March 2016

mZgAkl <a href="http://fbdnisdgysij.com/">fbdnisdgysij</a>, [url=http://jzowycxvfekb.com/]jzowycxvfekb[/url], [link=http://lkctxlgmquum.com/]lkctxlgmquum[/link], http://obyjjyzxsbxr.com/

Retrieved from "http://2008.igem.org/Team:Illinois/Antibody_Receptor_Tyrosine_Kinase_Fusion_Notebook"