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Illinois/30 July 2008
From 2008.igem.org
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.
