IGEM:University of Illinois Urbana Champaign/2009/Notebook/Bioware 2010 RNA Decoder/2010/06/30

iGEM Project name 1

Main project page Previous entry      Next entry

June 30, 2010 Today we tried cloning out MicA, MicF, OmpA, OmpF, and Hfq. The procedure we followed was: 10X Buffer 5.0 uL Pfu Turbo DNA Polymerase 0.4 uL Primer Forward 0.5 uL Primer Reverse 0.5 uL dNTP 1.0 uL Template (MG1655) 0.5 uL dH2O 42.1 uL Total= 50 uL A master mix instead of adding each of those amounts to each tube. The master mix was made as such (MM for 5 RXNs): 10X Buffer 25uL Pfu Turbo DNA Polymerase 2 uL dNTP 5 uL Template (MG1655) 2.5 uL dH2O 210.5 uL We also ran controsl for each reaction: one without template, and one without polymerase. In each water replaced the missing material. The PCR settings were: 95 C= 5 min. 95 C= 30 secs. 55 C= 30 secs. 72 C= 1 min. 72 C= 5 min. 4 C= Hold Steps 2-4 repeated 30 cycles We then ran a electrophoresis gel of my PCR results.