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
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