DNA extraction using DNeasy Plant Mini Kit. Preparatory work: 1. If using the kit for the first time, add ethanol to buffer AW and buffer AP3/E to obtain the working solutions. 2. Preheat a water bath to 65º C. Tissue disruption 1. Cut ~100mg young leaf tissue and place in a 2 ml Safe-Lock microtube containing two 5 mm stainless steel bead. 2. Freeze the tubes in liquid nitrogen for 30 s. 3. Place the tubes into the TissueLyser Adapter set and stabilize with clamps. Immediately grind the samples for 1 min at 30 Hz. 4. Disassemble the Adaptor set, remove the microtubes, and re-freeze the samples in liquid nitrogen for 30 s. 5. Repeat step 3, reversing position of microtubes within the Adaptor Set. DNA Preparation 1. Add 400 µl of buffer AP1 and 4 µl of RNase A stock solution to a maximum of 100 mg (wet weight) of ground tissue and vortex vigorously. 2. Incubate the mixture for 10 min at 65º C. Mix 2-3 times during incubation by inverting tube. 3. Add 130 µl buffer AP2 to the lysate, mix and incubate for 5 min on ice. Optional: Centrifuge the lysate for 5 min at 20,000x g (14,000 rpm). 4. Apply the lysate to the QIAshredder Mini Spin Column (lilac) placed in a 2 ml collection tube and centrifuge for 2 min at 20,000x g. 5. Transfer flow-through fraction from step 4 to a new tube without disturbing the cell debris pellet. 6. Add 1.5 volumes of Buffer AP3/E to the flow-through and mix by pipetting. 7. Apply 650 µl of the mixture from step 6, including any precipitate which may have formed, to the DNeasy Mini Spin Column sitting in a 2 ml collection tube. Centrifuge for 1 min at 6000x g (8000 rpm) and discard flow-through. 8. Repeat step 7 with remaining sample. Discard flow-through in the tube. 9. Place DNeasy Mini Spin Column in a new 2 ml collection tube, add 500 µl buffer AW to the Spin Column and centrifuge for 1 min at 6000x g (8000 rpm). Discard flow-through and reuse the collection tube in step 10. 10. Add 500 µl buffer AW to the Spin Column and centrifuge for 2 min at 20,000x g (14,000 rpm) to dry the membrane. 6
11. Transfer the column to a 2 ml microcentrifuge tube and pipette 100 µl of buffer AE directly onto the DNeasy membrane. Incubate for 5 min at room temperature (15-25 C) and then centrifuge for 1 min at 8000 rpm) to elute. 12. Repeat step 11 once DNA concentration check (Eppendorf biophotometer) Power on: choose the dsdna function Calibrate Biophotometer (100 µl distilled H 2 O or dilution buffer in a cuvette); Press Blank button. Press dilution function. a. Enter amount of DNA sample (2 µl), Press Enter. b. Enter amount of diluent (98 µl), Press Enter. Measure sample (2 µl DNA + 98 µl diluent). Press Sample button. Read the concentration on screen (output is printed). OD 260/280 can be used to determine purity of DNA. OD <1.8: proteins and other absorbers in sample (re-precipitate DNA) OD 1.8 2.0: absorption due to nucleic acids OD >2.0: chloroform or phenol contamination, re-precipitate with ethanol Using a pipette, remove sample from bottom corner of the cuvette, discard tip and reuse cuvette. DNA concentration, purity and quality check Prepare 0.8% agarose gel (e.g. 2.4 g agarose, to 300 ml 1 X TAE buffer). Microwave until boiling, cool while stirring using a magnet stirrer, add 15 µl (10 mg/ml) ethidium bromide (EtBr) for 300 ml gel, mix and cast the gel. (EtBr is a mutagen, wear gloves while handling) Prepare sample (2 µl DNA + 2 µl 5X Blue Juice dye + 6 µl distilled water). After gel is ready (45 mins), load sample. Load molecular mass ruler (e.g. from Biorad) Run at 40 volts for 10 minutes, then at 90-100 volts for 50 minutes. Visualize gel under UV light 7
a. Check concentration of samples through comparison with the molecular mass ruler. b. Check for presence of RNA (blob at the bottom). c. Check for presence of proteins (crown shape on DNA band) d. Check for quality of DNA (is it sheared?) by looking for smears. NB: Any of the above may negatively affect the PCR reaction. 8
PCR Protocol for M13 primers: A. Reaction mix*: 10x PCR Buffer 2.00 µl MgCl 2 (25 mm) 2.00 µl dntps (2 mm) 1.50 µl F primer (M13, 5 µm) 0.10 µl R primer (10 µm) 0.20 µl Dye M13 (10 µm) 0.20 µl Taq Gold 0.20 µl Nuclease free water 9.80 µl Total 16.00 µl Template DNA (10ng/µl) 4.0 µl Total reaction volume 20.0 µl * Work on ice B. PCR program 95ºC 10 min 95ºC - 30 sec; 57ºC (Tm of primer) - 45 sec; 72ºC - 45 sec: 30 cycles 95ºC - 30 sec; 53ºC - 45 sec; 72ºC - 45 sec: 10 cycles 72ºC 10 min NB: The PCR products will be used for running agarose gel (10 µl), polyacrylamide gel (5 µl) and capillary electrophoresis in ABI 3730 (5 µl). 9
Running Agarose gels: 2% (w/v) Prepare 2.0% agarose gel (e.g. 6.0 g agarose, to 300ml 1 X TBE buffer). Microwave until boiling. Let cool while stirring, using a magnet stirrer and stir bar. For 300 ml gel, add 15 µl (10mg/ml) ethidium bromide (EtBr), keep stirring. (EtBr is a mutagen, wear gloves while handling). Prepare gel box with tray, combs and end pieces*. Slowly pour Agarose when container is cool enough to touch. Let gel solidify for at least 45 minutes. Remove end pieces and add enough buffer to gel tank to cover the gel. Remove combs Prepare sample (mix 10 µl PCR product + 2 µl 5X Blue Juice dye). Load 10 µl of sample. Load Molecular mass standard. Hook up the electrophoresis apparatus to power supply. Run at 40 volts for 10 min, then at 160 volts for 90 min. Visualize EtBr-stained DNA in gels using a transilluminator or UV light box Take photograph and score gel. * Different gel systems differ in gel set up procedure. 10
Polyacrylamide gel electrophoresis Preparatory work: Clean plates in 5% NaOH, preferably overnight, or for at least 60 minutes if in a rush. Small Glass Plate Preparation: Clean small plate with 95% ethanol, Pipette 1ml of binding solution (50ml 95% EtOH + 250µl Acetic Acid + 70µl Bind Silane) onto plate, evenly spread with Kimwipe and allow to sit for 5-10 minutes. Complete three additional 95% ethanol rinses to remove excess binding solution. Large Glass Plate Preparation: Always change gloves before working on the large plate! Rinse with cold tap water, clean with Alconox if needed. Wash the large plate with 95% EtOH before applying Acryl-Glide or Sigma Cote. Dab a Kimwipe with Sigma Cote then evenly wipe (polish) over large plate. After 5-10 minutes, remove excess Sigma Cote by wiping the plate with a Kimwipe. Assemble Gel: Put spacers on the margin of the large plate with red rubber part at top. Place small plate, coated side down, on top of spacers on large plate. Align spacers and push red rubber spacer against small plate until it wrinkles. Place three metal clamps on each side. Pour Gel: Fill space between plates with gel mix and move back and forth. Push flat side of comb into gel until line in comb lines up with top of small plate. Place three clamps over the top plate and comb, let solidify for an hour. Gel stock solution (6%): 400g Urea + 167ml Acrylamide:Bis 19:1 mix + 100ml 10X TBE make vol. 1L with ddh 2 O Gel mix: 50ml of Gel solution + 45µl Temed + 300µl of 10% Ammonium Persulfate (APS) Gel Electrophoresis: Assemble the gel apparatus and pour 900ml 1X TBE buffer in top and bottom chambers, then gently with even pressure on both sides remove the comb. Connect apparatus to power supply and pre-run at 80 watts for at least 15 min. Add 5 µl polyacrylamide loading dye (24.25 ml Hi-Di Formamide + 500 µl 0.5M EDTA + 250 µl Dye) to the PCR product plate, give a quick spin. 11
Denature samples and ladder at 95º C for 5 minutes, place on ice immediately. Pipette buffer in and out of the gel well to remove any debris and set the comb. Load 5.0 µl sample into each well for 48/62 well-comb, or 3.0 µl for 94 well-comb. Run gel at 80 watts for 80 to 120 minute. Silver staining protocol Prepare Solutions: Fixing/Stop Solution (1 L): 900 ml Millipore Water + 100 ml acetic acid Staining Solution (1 L): 1 g silver nitrate to 1 L Millipore Water, mix well. Add 1.5ml of 37% Formaldehyde before use. Developer (1 L): 30 g Sodium Carbonate to 1 L of Millipore Water, store at 4º C. Add 200 µl sodium thiosulfate solution (0.01 g sodium thiosulfate to 1 ml of sterile ddh 2 O) and 1.5 ml of 37% formaldehyde before use. Separate the plates: After gel run, disassemble plates from the apparatus. Put the small plate face down and slowly remove the large plate by wedging a plastic scraper between them at the top. The gel should adhere to the small plate. Fix the gel: Place the gel plate up in the large plastic container and add all of Fixing/Stop solution. Shake @ 75 rpm for 20-30 minutes. Pour the Fixing/Stop solution back into the bottle for later use in terminating the developing reaction. Wash the gel: Rinse the gel 3 times (2-3 minutes each) with Millipore water @ 75 rpm. Stain the gel: Add the Staining solution to the container labeled for staining and place the plate in the container. Allow it to shake for 30 minutes @ 75 rpm. Rinse the gel: Dip the gel briefly into the tray containing Millipore Water, drain and place gel immediately into the tray of chilled developing solution. The time taken to dip the gel in the water and transfer it to developing solution should take no longer than 5-10 seconds. Develop the gel: Rock the gel by hand or @ 125 rpm until you see bands. Continue until bands are almost as dark as you want them (usually 3-5 minutes). 12
Stop development: To terminate the developing reaction and fix the gel, add ~1 L of Fixing/Stop solution directly to the developing solution. Incubate with shaking for 2-3 minutes. Longer incubation will fade the stain. Rinse the gel: Rinse the gel with 2 L of Millipore Water for 10 minutes or twice at 5 minutes each. Dry the gel: Take the plate out and air dry overnight. Fragment Analysis Using ABI3730 Preparatory work: Take out PCR reaction product and formamide solution from 4 o C. Prepare loading plates for ABI3730: Prepare loading buffer by mixing 10 µl of deionized formamide and 0.5 µl of GeneScan-500 LIZ internal size standard for each sample. A master mix should be made for multi-sample loading. Aliquot 10.5 µl loading buffer into 96-well sequencing plate. Transfer 3 µl PCR product into sequencing plate. Short spin the plate and cover with septa seal. Denature sample at 95 o C for 5 minutes in a thermal cycler. Chill samples on ice. Load plate to ABI3730 for analysis: Bring plate on ice to DNA core facility lab. 13
Short spin plate to bring samples to bottom of wells and then assemble plate into the loading cassette with same orientation (note: notch on the right up corner). Load plate into ABI3730. Start running and then collect data. Check genotyping data on GeneMapper: Open GeneMapper, load genotyping data. Modify Analysis Method setting. Set up panel for each data set. Analyze data, checking peaks for allele call. 14