Week 11. Semester 3. PCR on samples from April 2015

PCR was performed on samples collected 04/24/2015 and extracted 04/27/2015.
The following is the protocol utilized.

Polymerase Chain Reaction (11-18-15)

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

2μL DNA*and 10 μL Sterile Water (for positive controls)

12μL DNA and no water (for samples)

ThermoCycler at an annealing temperature of 55 ͦC for 34 cycles with hold at  4 ͦC

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

16s LEG-858: GTCAACTTATCGCGTTTGCT.

mip LpneuF: CCGATGCCACATCATTAGC (150 bp);

mip LpneuR: CCAATTGAGCGCCACTCATAG.

Samples collected 04/24/2015 coded 1-10 16s.

No optional step during extraction.

Extracted 04/27/2015

Sample No.	Location
1	Middle Football Irrigation Control Box (Biofilm)
2	South Gym Family Shower Drain (Biofilm)
3	Hacienda AC Roof Top (Biofilm)
4	DB-123 Station Z Eyewash Drain (Biofilm)
5	Center Field Football Sprinkler Head (Biofilm)
6	Football Snack Bar Fountain Head (Biofilm)
7	Hacienda Non-Potable (Biofilm)
8	North Valve Rooftop cooling Tower Sitting Water (Water)
9	F-Building East Spigot (Water)
10	Hacienda Non-Potable (Water)

Experimental Errors:

Used Master Mix from different tubes. 

PCR amplification. An illustration showing how DNA is amplified in the first four cycles. 

Courtesy of: West Coast Pathology Lab.