You will isolating Drosophila melanogaster DNA and setting up PCR reactions this week. These will be run overnight, and then frozen until after reading break. In addition, Will Hunter will be preparing a labelled probe from the plasmid pPBH (used previously in Lab 3). He will set up hybridization reactions using this probe and your Southern blots and plaque lifts from last week on Tuesday afternoon, and set up washes on Wednesday afternoon.
D. melanogaster genomic DNA prep
3 D. melanogaster (killed by freezing)
homogenization buffer: 100 mM Tris, pH 8.0
100 mM EDTA
1 % SDS
8 M potassium acetate (ice-cold)
isopropanol (room temperature)
70 % ethanol
ddH2O
1. Grind 3 flies in 100 µl of homogenization buffer in an Eppendorf tube.
2. Add 14 µl of ice-cold 8 M potassium acetate. Leave on wet ice for 15 minutes.
3. Spin in a microcentrifuge for 5 minutes.
4. Transfer the supernatant to a fresh tube.
5. Add 0.5 volumes of isopropanol to the supernatant, mix, and leave 5 minutes at room temperature.
6. Spin DNA pellet down (5 minutes, microcentrifuge).
7. Rinse the pellet with 70% ethanol, air dry, and redissolve in 300 µl of ddH2O.
You will be doing PCR reactions on DNA extracted from D. melanogaster. The primers used will be 2 pairs of primers specific for the mt:CoII gene (coding for the mitochondrial Cytochrome c oxidase subunit II) in the D. melanogaster mitochondrial genome. The first primer pair will be TLJ3034 and H3665; the second pair will be TLJ3034 and H3796. (The numbers for each primer refer to the position of the end of the primer on the D. melanogaster mitochondrial genome.) Each group that shares a bench should use one of these primer pairs, and the other group should use the other pair. When the primers are run on a gel, you will be able to compare the size of the fragments produced.
Each group will set up 2 reactions: an experimental reaction and a control reaction. Control reactions are important when doing PCR, as the high sensitivity of the system can easily amplify contaminating DNA. In order to pipette the small volumes needed more accurately, all ingredients except the DNA will be set up in one tube as a “master” reaction mix. 22 µl of this mix will then be transferred to a second tube. 3 µl of D. melanogaster DNA will be added to the experimental tube and 3 µl of ddH2O will be added to the control tube.
1 tube “master”
ddH2O 15 µl 30 µl
dNTP mix (1.5 mM) 1 µl 2 µl
10X Taq or PCR buffer 2.5 µl 5 µl
25 mM MgCl2 1.5 µl 3 µl
first primer 1 µl 2 µl
second primer 1 µl 2 µl
Taq polymerase 0.25 µl 0.5 µl
(Random primer extension – see also “Lab 5 references” in library)
boiling water bath (heat block, glass beaker, plastic floater, boiling chips)
37 C heat block, the kind that holds microcentrifuge tubes (ideally, screw-cap microcentrifuge tubes)
plasmid pPBH
Random Primed DNA Labelling Kit (Roche)
alpha 32P dCTP (3000 Ci/mMol)
TE pH 8.0
ProbeQuant G-50 Micro Columns (amersham pharmacia)
geiger counter, cafeteria tray, diaper material, large shield (for working behind), small shield (for transporting hot material to downstairs lab), 1 waste container for upstairs, 2 waste containers for downstairs (high level solid and high level aqueous)
several basics but will be labelled 'hot' – small beaker, P10, and P100, microcentrifuge, tip boxes and radioactive tape to label working area and all equipment
(2 probes will be prepared and pooled) – total reaction volume 20 µl for each
1. Heat ~ 100 ng plasmid DNA (in 9 µl ddH2O) to 99°C for 10 minutes to denature.
2. Snap cool on ice for 1 minute.
3. Add 1 µl each of 0.5 mM dATP, 0.5 mM dGTP, 0.5 mM dTTP.
4. Add 2 µl of reaction mixture (contains hexanucleotide primers, 10X reaction buffer.)
5. Add 5 µl of alpha 32P dCTP and 1 µl (2 Units) Klenow fragment
6. Incubate at 37°C for 90 minutes.
7. Stop reaction with 30 µl TE pH 8.0
8. Labelled Probe separated on ProbeQuant column.
9. 2 samples pooled and heated to to 99°C for 5 minutes to denature.
10. Snap cool on ice.
hydridization tubes
prehybridization solution: 50 mM sodium pyrophosphate
100 mM monobasic sodium phosphate
7 % SDS
100 µg/ml denatured salmon sperm DNA
Southern blots and plaque lifts placed in 25 ml prehybridization solution in 2 hydridization tubes. Rotated in hybridization oven at 68°C for 2 hours.
Denatured probe diluted to 2 ml in prehybridization solution and 1 ml added to each bottle. Hybridized overnight at 68°C.
washes
3X washes for 30-45 minutes each at 68°C. Filters exposed to film for varying times to obtain optimal exposure.