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 NEB Tm Calculator, as called for in the 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
Cas3: Touchdown PCR, Start Temp 63, -0.2 / cycle, Final Temp 57

Gel Results:

CasABCDE: Very faint bands near target length
Cas3: No bands

July 18, 2011

CasABCDE: Touchdown PCR, Start Temp 65, -0.2 / cycle, Final Temp 59

Gel Results:

CasABCDE: No bands

July 19, 2011

CasABCDE: Touchdown PCR, Start Temp 70, -0.2 / cycle, Final Temp 63
Cas3: Temp gradient with 3 rows from 72 to 55 (L to R: 55, 61.7, 71)
Gel Results:

CasABCDE: Clearer bands around target length, primer dimers

Cas3: Middle temperature was optimal, clear bands are visible (likely correct length, failed to run a hyperladder), primer dimers

July 20, 2011

CasABCDE: Touchdown PCR, Start Temp 71, -0.2 / cycle, Final Temp 64
Cas3: Gradient using tighter range around best temp from yesterday (65.6, 63.1, 61.2, 58.5)

Gel Results:

[[Image:]]
CasABCDE: Nonspecific, nothing usable, no bands in target range, dimerization [[Image:]]
Cas3: Small band at correct location, primer dimers

July 24, 2011

PCR of CasABCDE, Cas3 with new settings; Preparation for extraction, "PCRception" (PCRing the PCR results); Included an elongation step in each cycle, which was not done for the previous runs; Settings stored in PCR machine as ABCDE724, CAS3724

CasABCDE Settings and Results:
[[Image:]]
[[Image:]]
Nonspecific results, perhaps a hint of the correct bands in the second to last well
Cas3 Settings and Results:
[[Image:]]
[[Image:]]
Success for Cas3!! Extracted the band that lies around ~2500 (target is 2600). However, sequencing results were never conclusive and the sample was lost.

Primer Round 2