Team:Illinois/Antibody Receptor Tyrosine Kinase Fusion Notebook

From 2008.igem.org

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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 kit was used to extract the plasmids carrying the TE33 light and heavy chains.  From these plasmids we can run PCRs to amplify the regions of the antibody chains.
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DNA eluted in 100μL H2O and frozen.
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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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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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-
 
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-
 
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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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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
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-
|-
+
-
|mastermix
+
-
|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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|-
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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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-
 
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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
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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)
+
-
|0.5ul SCA link + 0.5ul flk link 2
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-
|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
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-
 
+
-
== 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
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-
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
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-
|1
+
-
|2
+
-
|3
+
-
|4
+
-
|5
+
-
|6
+
-
|-
+
-
|template
+
-
|colspan="4"|A=0.5uL  B=1.0uL  C=1.5uL
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-
|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
+
-
|}
+
-
 
+
-
== October 21, 2008 ==
+
-
 
+
-
Made 500ml LB media
+
-
500mL DI water
+
-
5g Tryptone
+
-
2.5g Yeast Extract
+
-
2.5g NaCl
+
-
Autoclaved on liquid setting, 15 min.
+
-
 
+
-
Frozen stocks of pSB1A7 and pSB1A3 from iGEM used to innoculate LB+Amp cultures for plasmid prep.
+
-
 
+
-
== October 22, 2008 ==
+
-
 
+
-
Cultures from yesterday used for plasmid prep (minispin kit)
+
-
 
+
-
restriction digests
+
-
{| class="wikitable" border="1"
+
-
|-
+
-
|tube
+
-
|1
+
-
|2
+
-
|3
+
-
|4
+
-
|5
+
-
|6
+
-
|7
+
-
|-
+
-
|5uL DNA
+
-
|pSB1A7
+
-
|pSB1A3
+
-
|H brick
+
-
|L brick
+
-
|SCA brick
+
-
|flk-1 brick
+
-
|flk-1 plasmid
+
-
|-
+
-
|5uL buffer2
+
-
|colspan="7"|
+
-
|-
+
-
|0.5uL BSA
+
-
|colspan="7"|
+
-
|-
+
-
|1uL RE 1
+
-
|colspan="7"|EcoRI
+
-
|-
+
-
|1uL RE 2
+
-
|colspan="6"|PstI
+
-
|HindIII
+
-
|-
+
-
|37.5uL H20
+
-
|colspan="6"|PstI
+
-
|HindIII
+
-
|-
+
-
|37.5uL H20
+
-
|colspan="7"|
+
-
|}
+
-
 
+
-
<!-- == 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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Retrieved from "http://2008.igem.org/Team:Illinois/Antibody_Receptor_Tyrosine_Kinase_Fusion_Notebook"