Team:Illinois/Antibody Receptor Tyrosine Kinase Fusion Notebook

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<!--PLACE TEXT BELOW HERE -->
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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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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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== July 19, 2008 ==
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Glycerol added to 10%, cells in freezer
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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 1μL 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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Plenty of colonies on all plates
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Also, making 5mL overnight cultures for mini-prep tomorrow.
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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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|DNA source
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|colspan="4"|mini-prep
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|colspan="4"|synthesized IDT genes
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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: 1. 94 degrees 5 min
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2. 94 degrees 1 min
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3. 50 degrees 1 min
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4. 72 degrees 1 min
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5. GOTO 2. 39 cycles
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6. 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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<!-- == 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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Latest revision as of 06:22, 29 March 2016

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