Handout, 446L/546L, 9/11/15
For purification of your PCR products (use qiaquick spin columns)
(LABEL YOUR COLUMNS/TUBES ACCURATELY!)
Protocol is on-line: http://biology.unm.edu/cmadema/4546/QIASpin.pdf
From that protocol
1). You will use the PCR reaction containing the amplicon for COI or 16S as indicated on the board.
There is 30 microliters of sample left (50-20 for gel). (LABEL YOUR COLUMNS/TUBES ACCURATELY!)
2). Add 500 μl of Buffer PB to the PCR sample and mix. It is not necessary to remove mineral oil or kerosene. For example,
add 500 μl of Buffer PB to 100 μl PCR sample (not including oil).
3). If pH Indicator I has been added to Buffer PB, check that the color of the mixture is yellow. If the color of the mixture is
orange or violet, add 10 μl of 3 M sodium acetate, pH 5.0, and mix. The color of the mixture will turn to yellow.
4). Place a QIAquick spin column in a provided 2 ml collection tube. (LABEL YOUR COLUMNS/TUBES ACCURATELY!)
5). To bind DNA, apply the sample to the QIAquick column and centrifuge for 30–60 s.
(LABEL YOUR COLUMNS/TUBES ACCURATELY!)
6). Discard flow-through. Place the QIAquick column back into the same tube. Collection tubes are re-used to reduce plastic
waste.
7). To wash, add 0.75 ml Buffer PE to the QIAquick column and centrifuge for 30–60 s.
8). Discard flow-through and place the QIAquick column back in the same tube. Centrifuge the column for an additional 1
min. IMPORTANT: Residual ethanol from Buffer PE will not be completely removed unless the flow-through is discarded before this
additional centrifugation.
Place QIAquick column in a clean 1.5 ml microcentrifuge tube. (LABEL YOUR COLUMNS/TUBES ACCURATELY!)
9). To elute DNA at increased DNA concentration, add 30 μl Buffer EB (10 mM Tris·Cl, pH 8.5) or water (pH 7.0–8.5) to the
center of the QIAquickmembrane for 1 minute and the centrifuge the column for 1min
IMPORTANT: Ensure that the elution buffer is dispensed directly onto the QIAquick membrane for complete elution of bound DNA.
The average eluate volume is 48 μl from 50 μl elution buffer volume, and 28 μl from 30 μl elution buffer.
Elution efficiency is dependent on pH. The maximum elution efficiency is achieved between pH 7.0 and 8.5.When using water, make
sure that the pH value is within this range, and store DNA at –20°C as DNA may degrade in the absence of a buffering agent. The
purified DNA can also be eluted in TE buffer (10mMTris·Cl, 1mMEDTA, pH 8.0), but the EDTA may inhibit subsequent enzymatic
reactions.
PERFORM sequencing reaction. BigDye v3.1 DyeTerminators.
Full protocol is on-line: http://biology.unm.edu/cmadema/4546/BDT31.pdf
(see over)
PERFORM sequencing reaction. BigDye v3.1 DyeTerminators.
Full protocol is on-line: http://biology.unm.edu/cmadema/4546/BDT31.pdf
Group
SAMPLE
1
2
3
5
6
7
8
9
10
1
2
3
5
4
5
4
1
2
TARGET/PRIMER
16S
COI
F
R
F
R
F,R
F
R
F
R
R
F
R
F
R
F
R
F
Per GROUP
1). Select your primers for the targets (16SF AND COIR or 16SR AND COIF, group 3 COIF and COIR, See table above), 1 tube each
of forward primer and of the reverse primer.
2). Dilute each primer 30x with milliQ water. The tube contains 2 μl of primer at 50 μmolar.
CHECK WITH INSTRUCTOR WHEN IN DOUBT
3). Use the same P20 to make and to aliquot a master mix: 2 μl BIGDYE, 4 μl 5x buffer, 4 μl milliQ water
4) divide your mastermix: put 5 μl in each of 2 PCR tubes.
LABEL!!! Group number, target amplicon (16S or COI) seqeucne and F or R (e.g 1F and 1R).
5). Add 4 μl of purified amplicon, MAKE SURE TO PUT 16S in the 16S tube AND COI in the COI tube
6). Add 1 μl of diluted forward primer to the F labeled sequencing reaction, and 1 μl of diluted reverse primer to the R
labeled sequencing reaction.
DO NOT put both primers in one tube!!
MAKE SURE TO PUT 16S in the 16S tube AND COI in the COI tube
7). Spin and place tubes in collection rack/thermal cycler for thermal cycling.
CLEAN-UP sequencing reactions.
DO NOT DISTURB THE PELLET! These are your sequencing products.
1). Set up two 1.7ml eppendorf tubes, labeled group number, target and F or R and add 100 μl 100% EtOH and 4 μl 3M NaAc.
Transfer sequencing reactions from the PCR tubes to the correctly labeled tubes. Invert and spin 10’ max RPM at
roomtemperature.
2). Discard supernate, rinse pellet with 400 μl 75% EtOH, spin 5’ max RPM at roomtemperature.
3). Discard supernate, take out last few drops, dry to air, give to instructor for reading of extension products.