Illinois/30 July 2008

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(New page: '''Antibody/GPCR Fusion''' *Autoclaved Glass Beads and Eppendorf tubes)
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'''LAB WORK'''
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'''Antibody/GPCR Fusion'''
'''Antibody/GPCR Fusion'''
*Autoclaved Glass Beads and Eppendorf tubes
*Autoclaved Glass Beads and Eppendorf tubes
 +
 +
'''Receptor Tyrosine Kinase method'''
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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
 +
|2
 +
|3
 +
|4
 +
|5
 +
|6
 +
|7
 +
|8
 +
|-
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|Antibody chain
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|H
 +
|H
 +
|L
 +
|L
 +
|H
 +
|H
 +
|L
 +
|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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|-
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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
 +
|5ul
 +
|3ul
 +
|5ul
 +
|3ul
 +
|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: 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.
 +
[[Image:07-30-08_AbFrag_Gel1.jpg|none]]

Latest revision as of 04:03, 23 September 2008

LAB WORK

Antibody/GPCR Fusion

  • Autoclaved Glass Beads and Eppendorf tubes

Receptor Tyrosine Kinase method

Assembly PCR: amplify antibody gene fragments and then assemble them, light and heavy chains

tube 1 2 3 4 5 6 7 8
Antibody chain H H L L H H L L
DNA source mini-prep synthesized IDT genes
template 0.5ul
primers 1ul of appropriate forward and reverse primer
MgCl2 3ul 5ul 3ul 5ul 3ul 5ul 3ul 5ul
master mix 20ul
H20 to 50ul total volume

Master mix already contains MgCl2; should have added extra to some tubes.

PCR program: 1. 94 degrees 5 min 2. 94 degrees 1 min 3. 50 degrees 1 min 4. 72 degrees 1 min 5. GOTO 2. 39 cycles 6. HOLD at 4 degrees

1.5% agarose gel made, 0.75g agarose in 50ml 0.5X TBE w/10ul EtBr.

20ul of PCR produts loaded on gel w/4ul 6X loading buffer, run at 200V for about half an hour.

All lanes had lots of DNA at ~375bp, PCR worked.

07-30-08 AbFrag Gel1.jpg
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