PCR Primers used by Aguirre Lab: (last updated November 4, 2021)


PROTOCOLS:

Below are some of the common protocols and analytical methods used in our lab:

Genomics:

Quibit 4 Broad Spectrum Assay Protocol. Protocol for using the Qubit Broad Spectrum Assay to quantify DNA for genomic applications.

Genotyping:

Spreadsheet Program for Primer Preparation. This is a spreadsheet that calculates appropriate volumes of liquid to add to Invitrogen primers to prepare concentrated primer stocks. Input primer ug's and Molecular Weight (ug/umole) values from Invitrogen data sheets and the program gives you the volume of TE (Tris-EDTA) in ul to add to achieve a concentration of 100umol. These concentrated stocks are then diluted 1:10 in water to prepare working stocks for PCR.

Phenol-Chloroform DNA Extraction Protocol. Our standard phenol-chloroform based DNA extraction method for fish tissue modified from a protocol used by David Kingsley's lab at Stanford University in the 2000's. We primarily use fin clips and our tissues are preserved in 95% ethanol or forzen prior to DNA extraction. Updated in June 2019 by Roberto Cucalon to include an optional RNase step to eliminate RNA for genomic applications.

Chelex DNA Extraction Protocol for Insect Tissue. A chelex based DNA extraction protocol for insect tissue.

Miniprep Protocol. Miniprep protocol for isolating plasmids after bacterial transformation (molecular cloning). Modified from "Short Protocols in Biology".

Microsatellite Multiplex PCR Protocol (Qiagen). Multiplex PCR: This is a protocol for multiplex PCR of stickleback microsatellite loci using Qiagen kits. The details were worked out by Cory Drevecky (M.S. student).

RAD-Seq Library Preparation protocol. Modified from baddna.uge.edu by Roberto Cucalon.

Guide for running Barcode Generator program. Generates individual barcodes for individual identification of specimens in multiplex genomic sequencing applications.

Reagents for Genotyping:

TE Buffer Preparation Protocol. DNA storage: Protocol for preparing the TE (Tris-EDTA) Buffer that we use for long term storage of concentrated stock DNA.

0.5X TBE Buffer Preparation Protocol. Gel electrophoresis: Protocol for preparing the 0.5X TBE Buffer that we use for agarose gel electrophoresis.

DNA Ladder Preparation Protocol. Gel electrophoresis: Protocol for preparing the DNA ladder that we use for our agarose gels. The ladder is from New England Biolabs.

SYBR-Safe Preparation Protocol. Gel electrophoresis: Protocol for preparing SYBR-Safe to visualize DNA under UV light in agarose gel electrophoresis. SYBR-Safe is a great substitute for ethidium bromide.

EXOSAP-IT Protocol. DNA sequencing: Protocol for using EXOSAP-IT to purify PCR products for direct sequencing. EXOSAP-IT is a great substitute for PCR purification kits when you get clean products. It has worked great for us.

Agar Plate Preparation. LB/Ampicillin Agar Plate Preparation for Molecular Cloning.

Development:

Lab embryo collection sheet for Astyanax developmental series.

PBS Preparation Protocol. General Lab Reagent: We use phosphate buffered saline (PBS) for our 4% paraformaldehyde fixative (for fixing specimens for in situ hybridization) and as a general reagent in the lab.

4% Paraformaldehyde Fixative Preparation Protocol. Fixative for in situ hybridization: This 4% paraformaldehyde fixative (PFA) is used to fix specimens for in situ hybridization.

Insitu Hybridization Chain Reaction Protocol, Version 3. Protocol for Insitu staining of vertebrate embryos that magnifies target signal and allows simultaneous mapping of multiple targets. Modified from Choi et al. (2014). This protocol is for version 3 of the method. Updated October 31, 2017.

DAPI Protocol. Protocol for preparing and using DAPI solution for staining nucleic acids in preserved embryos.

Morphology/Fish:

Alizarin Staining Protocol. Protocol for staining external bone red in stickleback fish.

MS-222 Preparation Protocol (for euthanasia). Protocol for making MS-222 for use as a euthanasia agent.

Data Analysis:

Allometric size adjustment of linear measures. Simple method for size-adjusting linear measures based on Reist (1985).

PHYLIP Tree Protocol. Protocol for making Neighbor-joining trees from morphological or genetic distance matrices in PHYLIP.

Arlequin Formatting. Instructions for formatting sequence data for use in Arlequin.

Network Formatting. Instructions for formatting sequence data for use in Network software from Flexus-engineering.com.


Last updated: November 4, 2021