Week 2. Semester 3. PCR on positive controls and Analysis

Last Friday, September 11^th, we set up a PCR on all our positive controls from the summer. Before we can collect new samples for DNA extraction and analysis, we must make sure our positive controls preserved well in the break and that we have no contamination. Once we collect samples we will compare the banding pattern of the amplified genes (16s and mip) against our positive controls, so it is imperative the controls are nice and clean and band appropriately. 

The PCR was prepared as follows:

PCR Tube: 20 μL MasterMix, 4 μL Forward/Reverse Primers,

1μL DNA*and 11 μL Sterile Water

ThermoCycler at an annealing temperature of 55 ͦC for 34 cycles.

Primers Used:  16s LEG-226: AAGATTAGCCTGCGTCCGAT (654 bp);

16s LEG-858: GTCAACTTATCGCGTTTGCT.

mip LpneuF: CCGATGCCACATCATTAGC (150 bp);

mip LpneuR: CCAATTGAGCGCCACTCATAG.

Banding pattern expected for the two amplified genes (16s in center of gel and mip in right).

 Picture courtesy of the Spring 2015 Legionella team.