IGEM:Missouri University of Science and Technology/2009/Notebook/Cyanobacteria Mediated Filtration of Coal Flue/2013/07/31

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Date: 7/31/13 People in lab: Emily Puleo, Kelsey Crossen

Title: PCR of norCB, dilution of norCB primers

Start Time: 4:57 PM

Purpose: PCR norCB from P. aeruginosa, adding iGEM prefix and suffix; to prepare primers for future lab work

Protocol: LTM ed. 2 pg. 48 Exceptions: 1. Less than 0.1 uM final primer concentration.


Sample Label Description Source Label Quantity
7/31/13 A norCB 1 Amplified norCB, with prefix/suffix, 7.5uL template P. aeruginosa 7/30 1
7/31/13 A norCB 2 Amplified norCB, with prefix/suffix, 5.63uL template P. aeruginosa 7/30 1
7/31/13 A norCB 3 Amplified norCB, with prefix/suffix, 3.75uL template P. aeruginosa 7/30 1
7/31/13 norCB F Forward primer for norCB, 0.73 uM norCB Forward Primer 1
7/31/13 norCB R Reverse primer for norCB, 0.87 uM norCB Reverse Primer 1

Results: Sample 3 had too little product and was discarded.

Notes: Different amounts of template used to find optimum amount needed.

Forward primer: (24.2*10^-9)/(0.33*10^-3) mol/L = 73 uM, (73uM)(10uL)=(0.73uM)V2, V2 = 1000uL

Reverse primer: (28.7*10^-9)/(0.33*10^-3) mol/L = 87 uM, (87uM)(10uL)=(0.87uM)V2, V2 = 1000uL

Used Dr. Shannon's thermocycler, copied "igem_pcr" program

Stop Time: 9:00 PM

Next: Run samples on a gel, extract the stronger band from between them, sequence the result.