Team:Arizona State/Notebook/PCRLog

Overview

This logbook is a record of the majority of our attempts to PCR amplify CRISPR-associated (Cas) ABCDE and Cas 3 of E. coli K12 MG1655. The record begins after our attempt to individually PCR amplify each of the 6 genes, which was unsuccessful. Here, three different sets of primers were used to attempt PCR amplification of the Cas genes in two sections.

Notes

Suggested annealing temperatures are based on [http://www.neb.com/nebecomm/tech_reference/TmCalc/Default.asp NEB Tm Calculator], as called for in the [http://www.neb.com/nebecomm/tech_reference/polymerases/phusion_high.asp?&utm_source=Google&utm_medium=CPC&utm_term=+phusion&utm_campaign=Phusion NEB Phusion DNA Polymerase] protocol.

Desired band for CasABCDE at ~4300bp

Desired band for Cas3 at ~2667bp

Primer Round 1

July 17, 2011

CasABCDE: Touchdown PCR, Start Temp 70, -0.2 / cycle, Final Temp 64</p>
Cas3: Touchdown PCR, Start Temp 63, -0.2 / cycle, Final Temp 57</p>

Gel Results:

[[Image:]]

CasABCDE: Very faint bands near target length

Cas3: No bands

Primer Round 2