July 19, 2010
We redid the PCR's from Saturday. The primers were not diluted to 1/10 their original concentrations.
- RXN 1 (MicA)
- 10 uL 10X Pfu Buffer
- 0.4 uL Pfr DNA Polymerase
- 0.4 uL Primer Forward
- 0.4 uL Primer Reverse
- 1.0 uL 4X dNTP
- 10 uL MG1655 Template
- 32.8 uL dH2O
- Total= 50 uL
- RXN 2 (OmpA)
- 10 uL 10X Pfu Buffer
- 0.4 uL Pfr DNA Polymerase
- 0.4 uL Primer Forward
- 0.4 uL Primer Reverse
- 1.0 uL 4X dNTP
- 10 uL MG1655 Template
- 32.8 uL dH2O
- Total= 50 uL
- RXN 3 (MicF):
- 10 uL 10X Pfu Buffer
- 0.4 uL Pfr DNA Polymerase
- 0.4 uL Primer Forward
- 0.4 uL Primer Reverse
- 1.0 uL 4X dNTP
- 10 uL MG1655 Template
- 32.8 uL dH2O
- Total= 50 uL
- RXN 4 (OmpF)
- 10 uL 10X Pfu Buffer
- 0.4 uL Pfr DNA Polymerase
- 0.4 uL Primer Forward
- 0.4 uL Primer Reverse
- 1.0 uL 4X dNTP
- 10 uL MG1655 Template
- 32.8 uL dH2O
- Total= 50 uL
- RXN 5 (GadY)
- 10 uL 10X Pfu Buffer
- 0.4 uL Pfr DNA Polymerase
- 0.4 uL Primer Forward
- 0.4 uL Primer Reverse
- 1.0 uL 4X dNTP
- 10 uL MG1655 Template
- 32.8 uL dH2O
- Total= 50 uL
- RXN 6 (GadX)
- 10 uL 10X Pfu Buffer
- 0.4 uL Pfr DNA Polymerase
- 0.4 uL Primer Forward
- 0.4 uL Primer Reverse
- 1.0 uL 4X dNTP
- 10 uL MG1655 Template
- 32.8 uL dH2O
- Total= 50 uL
- RXN 7(Hfq)
- 10 uL 10X Pfu Buffer
- 0.4 uL Pfr DNA Polymerase
- 0.4 uL Primer Forward
- 0.4 uL Primer Reverse
- 1.0 uL 4X dNTP
- 10 uL MG1655 Template
- 32.8 uL dH2O
- Total= 50 uL
- RXN 8 (EGFP)
- 10 uL 10X Pfu Buffer
- 0.4 uL Pfr DNA Polymerase
- 0.4 uL Primer Forward
- 0.4 uL Primer Reverse
- 1.0 uL 4X dNTP
- 1 uL PXG-10 Template
- 41.8 uL dH2O
- Total= 50 uL
- RXN 9 (Polymerase Control)
- 10 uL 10X Pfu Buffer
- 0.4 uL Primer Forward
- 0.4 uL Primer Reverse
- 1.0 uL 4X dNTP
- 10 uL MG1655 Template
- 33.2 uL dH2O
- Total= 50 uL
- RXN 10 (Template Control)
- 10 uL 10X Pfu Buffer
- 0.4 uL Pfr DNA Polymerase
- 0.4 uL Primer Forward
- 0.4 uL Primer Reverse
- 1.0 uL 4X dNTP
- 42.8 uL dH2O
- Total= 50 uL
- RXN 11 (Primer Control)
- 10 uL 10X Pfu Buffer
- 0.4 uL Pfr DNA Polymerase
- 1.0 uL 4X dNTP
- 10 uL MG1655 Template
- 33.6 uL dH2O
- Total= 50 uL
The PCR setting that was used:
- 95 C: 5 min.
- 95 C: 30 sec.
- 51 C: 30 sec.
- 72 C: 1.5 min.
- 72 C: 10 min.
- 4 C: hold
Numbers 2-4 were repeated 32 times.
The Concentrations of the templates were:
- PXG-10: 475 ng/uL
- MG1655: 2.8 ng/uL
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