Download DNA Extraction, PCR Amplification and Sequencing: the IGS

SUPPLEMENT: DNA Extraction, PCR Amplification and Sequencing: the IGS
Locus
DNA Extraction: Approximately 50 mg of dried or lyophilized gill tissues, or slices of
tissues preserved in CTAB buffer, were placed in 2.0 ml microcentrifuge tubes with 4-5 3
mm glass beads and macerated using a Mini-BeadBeater-8 (BioSpec Products Inc.,
Bartlesville, OK) set at 3/4 speed for one minute (100 ml of 2X CTAB is 10 ml of a 1 M
stock of TRIS-HCL pH 8.0, 28 ml of a 5 M stock of NaCl, 4 ml of a 0.5 M stock of
EDTA, 2 g of CTAB and 54 ml of dH2O). Genomic DNA was extracted using a Qiagen
DNeasy Tissue kit (Qiagen Inc.,Germantown, MD), according to manufacturer’s
specifications with the following modifications: incubation times with lysis buffer
(Buffer ATL) were extended to one hour; after incubation, the tubes were spun in a
microcentrifuge to pellet cellular debris at the bottom, and the liquid was removed to a
new tube; incubation with buffer AE was extended to five minutes, and the final volume
eluted was 200 ul. To avoid cross-contamination among samples equipment was washed
with a dilute bleach solution after every round of extractions, dedicated equipment and
filtered tips were used for all extraction procedures, and extraction blanks were extracted
along with samples. The quantity and purity of genomic DNA was determined using a
NanodropTM Spectrophotometer (NanoDrop Technologies, Wilmington, DE).
PCR, cloning, and sequencing parameters: All PCR products were obtained with the
following recipe: 2.5 µl of 10X PCR buffer (containing 15 mM MgCl2), 2 µl of 2.5 mM
each of all four dNTPs, 1 µl of each primer (10 µM), and 0.1 µl of Platinum Taq
(Invitrogen) (5 U/µl); reactions were brought to a final volume of 25 µl with water.
Because of the length of the IGS region, and the potential for secondary structures, a PCR
enhancer (Ralser et al., 2006) containing a final concentration of 0.54 M betaine, 1.34
mM DL-Dithiothreitol (DTT), 1.34% Dimethyl Sulfoxide (DMSO), and 11 ug/ml Bovine
Serum Albumin (BSA) was used in each reaction. PCR reactions were amplified in a
BioRad iCycler with the following parameters: 5 minutes at 95˚C, followed by 35 cycles
of one minute at 95˚C, one minute 50˚C, and two minutes at 72˚C, and a final elongation
of 7 minutes at 72˚C.
All PCR products were cleaned using the Promega Wizard Geneclean kit (Promega
Corporation, Madison, WI), following manufacturer’s specifications. Sequences were
obtained using an Applied Biosystems 3730 sequencer. All sequence traces were edited
using the program Sequencher v. 4. (Gene Codes Corporation, Ann Arbor, MI).
LITERATURE
 Ralser M, Querfurth R, Warnatz HJ, et al. (2006) An efficient and economic
enhancer mix for PCR. Biochemical and Biophysical Research Communications,
347, 747-751.