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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== 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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== 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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== July 18, 2008 ==
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We made competent cells to use for transformations.
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Procedure:
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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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== July 19, 2008 ==
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Transformation: We received the synthesized TE33 antibody genes from IDT on plasmids.  We transformed these plasmids into DH5a cells and plated on LB+amp the find transformants.
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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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== July 21, 2008 ==
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Observations:
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There were plenty of transformants on the plates.  5ml of LB+amp was inoculated for plasmid extraction the next day.
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== 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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== 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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== 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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|-
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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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|-
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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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|-
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|primers
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|colspan="8"|1ul of appropriate forward and reverse primer
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|-
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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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|-
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|master mix
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|colspan="8"|20ul
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|-
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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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== 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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|-
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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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|-
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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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|-
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|template
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|colspan="4"|0.5ul of both heavy and light chains
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|-
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|primers
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|colspan="8"|1ul of appropriate forward and reverse primer
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|-
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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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|-
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|master mix
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|colspan="4"|20ul
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|-
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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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-
 
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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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== 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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|-
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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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|-
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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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|-
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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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|-
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|master mix
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|colspan="5"|20ul
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|-
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|H20
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|colspan="5"|to 50ul total volume
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-
|}
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Annealing temps: 40, 43.8, 50, 54, 60, and 64 degrees
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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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# 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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-
 
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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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== 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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== 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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== 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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== 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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== 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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|-
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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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|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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|-
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|
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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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|-
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|primers
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|colspan="8"|1uL forward & reverse for all
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|-
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|mastermix
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-
|colspan="8"|8.25uL for all
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-
|-
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|H2O
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-
|colspan="8"|39.25uL for all
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-
|}
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-
 
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{| class="wikitable" border="1"
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|-
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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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|-
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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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|-
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|
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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
+
-
|-
+
-
|mastermix
+
-
|colspan="8"|8.25uL for all
+
-
|-
+
-
|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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-
|-
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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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|-
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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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-
|
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|A
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-
|B
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-
|A
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-
|B
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-
|-
+
-
|primers
+
-
|colspan="8"|1uL forward & reverse for all
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-
|-
+
-
|mastermix
+
-
|colspan="8"|8.25uL for all
+
-
|-
+
-
|H2O
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-
|colspan="8"|39.25uL for all
+
-
|}
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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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-
 
+
-
== 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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-
 
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-
PCR to assemble SCA-flk-1 fusion and increase flk-1 yield
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-
 
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{| class="wikitable" border="1"
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|-
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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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|-
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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
+
-
|0.5ul SCA link + 0.5ul flk link 1 (crude)
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-
|0.5ul SCA link + 2ul flk link 1
+
-
|0.5ul SCA link + 2ul flk link 1 (crude)
+
-
|0.5ul SCA link + 0.5ul flk link 2
+
-
|0.5ul SCA link + 0.5ul flk link 2 (crude)
+
-
|0.5ul SCA link + 2ul flk link 2
+
-
|0.5ul SCA link + 2ul flk link 2 (crude
+
-
|0.5ul SCA link + 0.5ul flk link 3
+
-
|0.5ul SCA link + 0.5ul flk link 3 (crude)
+
-
|0.5ul SCA link + 2ul flk link 3
+
-
|0.5ul SCA link + 2ul flk link 3 (crude)
+
-
|-
+
-
|primers
+
-
|colspan="20"|1uL forward & reverse for all
+
-
|-
+
-
|mastermix
+
-
|colspan="20"|8.25uL for all
+
-
|-
+
-
|H2O
+
-
|colspan="20"|to 50ul total volume
+
-
|}
+
-
 
+
-
PCR program
+
-
# 94 degrees 4 min
+
-
# 94 degrees 1 min
+
-
# 50 degrees 1 min
+
-
# 72 degrees 3 min
+
-
# GOTO 2. 29 cycles
+
-
# HOLD at 4 degrees
+
-
 
+
-
== September 27, 2008 ==
+
-
 
+
-
*0.8% agarose gel run of products from yesterday, 200V for ~45 minutes
+
-
<br>
+
-
[[Image:9_27_08.jpg|none]]
+
-
 
+
-
*first 8 reactions may have worked but bands not great, assembly reactions did not work
+
-
 
+
-
== September 28, 2008 ==
+
-
 
+
-
0.8% gel run of yesterday's products, 150 V, 38 min, 200 V, 10 min
+
-
 
+
-
== September 30, 2008 ==
+
-
 
+
-
p1010 plasmid for biobicks cut out of plate 1003, well 4A; soacked in 5uL warm TE (50°C) for ~20 min, heat shocked at 50°C for 60s, placed on ice 2 min, 200uL LB added, incubated at 37°C, 1 hour, plated on LB + Amp and incubated overnight.
+
-
 
+
-
Restriction digest of our biobricks
+
-
18uL H2O
+
-
2uL 10x buffer Z
+
-
1uL EcoRI and PstI
+
-
1,2,4 uL DNA (H chain, L chain, SCA, flk)
+
-
 
+
-
p1010 transformatoin was stupid, transformed another plasmid instead, 1008, 1C
+
-
+
-
{| class="wikitable" border="1"
+
-
|-
+
-
|tube
+
-
|1
+
-
|2
+
-
|3
+
-
|4
+
-
|5
+
-
|6
+
-
|-
+
-
|template
+
-
|colspan="4"|A=0.5uL  B=1.0uL  C=1.5uL
+
-
|colspan="2"|(all flk-1 B)
+
-
|-
+
-
|primers
+
-
|colspan="8"|1uL forward & reverse for all
+
-
|-
+
-
|mastermix
+
-
|colspan="8"|8.25uL for all
+
-
|-
+
-
|H2O
+
-
|colspan="2"|39.25uL for A
+
-
|colspan="2"|38.75uL for B
+
-
|colspan="2"|38.25uL for C
+
-
|-
+
-
|colspan="7"|
+
-
|-
+
-
|tube
+
-
|1
+
-
|2
+
-
|3
+
-
|4
+
-
|5
+
-
|6
+
-
|-
+
-
|gradient
+
-
|55.0
+
-
|57.8
+
-
|59.3
+
-
|61.0
+
-
|63.5
+
-
|65.0
+
-
|-
+
-
|temps
+
-
|(1)
+
-
|(5)
+
-
|(6)
+
-
|(7)
+
-
|(9)
+
-
|(12)
+
-
|}
+
-
Run for 39 cycles
+
-
 
+
-
== October 2, 2008 ==
+
-
 
+
-
PCR products run on 0.8% gel, 200V, ~40min
+
-
Same results as always
+
-
 
+
-
pSB1A3 & pSB1A7 cut out of registry and transformed
+
-
Plated on LB+Amp
+
-
 
+
-
== October 3, 2008 ==
+
-
 
+
-
PCR from yesterday may have kind of worked, just not a lot of product, running another gel to find out
+
-
 
+
-
Transformation failed, transforming w/ RDR plasmid to test transformation protocol
+
-
 
+
-
Hard to tell if PCR products are correct length, so-so bands
+
-
 
+
-
== October 8, 2008 ==
+
-
Transformations to get plasmids 1003:2F, 1008:10D, 1008:1C, 1008:6G
+
-
 
+
-
== October 10, 2008 ==
+
-
DNA purification using S prime PerfectPrep(TM) Spin Mini kit on transformed bacteria from 10/8/2008
+
-
 
+
-
== October 14, 2008 ==
+
-
Transformation using plasmid from 10/10/2008
+
-
 
+
-
== October 16, 2008 ==
+
-
Transformation failed, duh
+
-
top 10
+
-
50uL cells transformed with 1003:10D, 1003:2F, plasmid from 10/10/2008, and pUC19 control
+
-
 
+
-
PCR to make ARTK fusion
+
-
 
+
-
{| class="wikitable" border="1"
+
-
|-
+
-
|tube
+
-
|1
+
-
|2
+
-
|3
+
-
|4
+
-
|5
+
-
|-
+
-
|template
+
-
|0.5uL SCA + 0.5uL flk-link3(8)
+
-
|0.5uL SCA + 1.0uL flk-link3(8)
+
-
|0.5uL SCA + 1.5uL flk-link3(8)
+
-
|0.5uL SCA + 2.0uL flk-link3(8)
+
-
|0.5uL SCA + 1.5uL flk-link3(8)
+
-
|-
+
-
|primers
+
-
|colspan="4"|1.0uL forward & reverse
+
-
|none
+
-
|-
+
-
|mastermix
+
-
|colspan="5"|20.0uL for all
+
-
|-
+
-
|H2O
+
-
|27.0uL
+
-
|26.5uL
+
-
|26.0uL
+
-
|25.5uL
+
-
|28.0uL
+
-
|}
+
-
 
+
-
== October 17, 2008 ==
+
-
 
+
-
Transformation failed. 
+
-
1% gel of yesterday's PCR run, 200V, ~40min, no bands
+
-
 
+
-
== October 18, 2008 ==
+
-
 
+
-
Using phusion to get ARTK
+
-
 
+
-
{| class="wikitable" border="1"
+
-
|-
+
-
|tube
+
-
|1
+
-
|2
+
-
|3
+
-
|4
+
-
|5
+
-
|-
+
-
|template
+
-
|0.5uL SCA + 0.5uL flk-link3(8)
+
-
|0.5uL SCA + 1.0uL flk-link3(8)
+
-
|0.5uL SCA + 1.5uL flk-link3(8)
+
-
|0.5uL SCA + 2.0uL flk-link3(8)
+
-
|0.5uL SCA + 1.5uL flk-link3(8)
+
-
|-
+
-
|primers
+
-
|colspan="4"|1.0uL forward & reverse
+
-
|no primers
+
-
|-
+
-
|buffer
+
-
|colspan="5"|10.0uL for all
+
-
|-
+
-
|phu
+
-
|colspan="5"|0.5uL for all
+
-
|-
+
-
|H2O
+
-
|36.5uL
+
-
|36.0uL
+
-
|35.5uL
+
-
|35.0uL
+
-
|37.5uL
+
-
|}
+
-
 
+
-
<!-- == Insert Date Here ==
+
-
* lab procedure
+
-
** more lab procedure
+
-
* second lab procedure
+
-
** more about second lab procedure
+
-
COPY/PASTE THEN REMOVE THIS AND BRACKETS-->
+
-
 
+
-
{{bottom_template}}
+

Latest revision as of 06:22, 29 March 2016

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