Laboratory Protocol Notebook
1
Lab Notebook
1.1
Important links:
2
Goals and Expectations
3
Isolation and Culturing
3.1
Culturing
3.2
Isolation From Fresh Poop
3.3
Liquid Sporulation Culture
4
Poop Extractions
4.1
Fecal Pellet Storage:
4.1.1
Notes Before Beginning:
4.2
DNA Extraction Protocol:
5
Water Extraction Protocol for Sterivex Filters
5.1
Hot Lysis
5.2
Phenol/Chloroform Extraction
5.3
Ethanol Precipitation
5.4
Solutions To Prepare
5.4.1
DNA Extraction Buffer (DEB) Makes 45mL
5.4.2
Low EDTA TE:
5.4.3
3 M sodium acetate, pH 5.2
5.4.4
5 N NaCl
5.4.5
NaOH (2N)
6
Soil Extractions
6.1
Detailed Procedure from DNeasy Powersoil Extraction kit
7
High Molecular Weight DNA Extraction
7.1
Protocol
7.2
Bead-cleaning protocols
7.3
Required PPE
7.4
Pre-Extraction Steps
7.5
Critical Safety Information.
7.6
Reagent Function and Troubleshooting
7.7
References
8
PCR Protocols
8.1
DreamTaq (Any Primers)
8.1.1
Protocol Mixture (DreamTaq)
8.1.2
Protocol Conditions (DreamTaq)
8.2
ITS Metabarcoding (ITS1-F/ITS2)
8.2.1
Protocol Mixture (ITS1-F/ITS2)
8.2.2
Protocol Conditions (ITS1-F/ITS2)
8.3
ITS1/ITS4
8.3.1
Protocol Mixture (ITS1/ITS4)
8.3.2
Protocol Conditions (ITS1/ITS4)
8.4
LSU
8.4.1
Protocol Mixture (LSU)
8.4.2
Protocol Conditions (LSU)
8.5
BASF611/BASR1340
8.5.1
Protocol Mixture (BAS)
8.5.2
Protocol Conditions (BAS)
8.6
EXOSAP
8.6.1
Protocol Mixture (EXOSAP)
8.6.2
Protocol Conditions (EXOSAP)
8.7
Primer sequences
8.7.1
16s for bacteria
8.7.2
LSU
8.7.3
ITS1 (metabarcoding)
8.7.4
ITS whole region
9
A basic roadmap of the cluster
9.1
How to log in
10
Safety and PPE
11
Submitting samples to Novogene
12
Culture-Enriched Mycobiome Protocol for Amphibian Feces
12.1
Written by Javier Tabima, Tested by Aris Papadimitrou and Sam Matthews
12.2
Purpose:
12.3
Overview
12.4
Materials
12.5
Enrichment Medium (Generalist)
12.6
Inoculation
12.7
Incubation
12.8
Harvesting the Enrichment
12.9
DNA Extraction
12.10
ITS Barcoding / Amplicon Sequencing
12.11
Optional Modifications
12.12
10. Expected Outcomes
Tabima Lab
Laboratory Notebook
Chapter 10
Safety and PPE
(No accidents allowed till I finish this chapter. LPC 10/14/2022)