Guidelines for Using the Sage Science Pippin Pulse

Transcription

1 Please note: the shared protocols described herein may not have been validated by Pacific Biosciences and are provided as-is and without any warranty. Use of these protocols is offered to those customers who understand and accept the associated terms and conditions and wish to take advantage of their potential to help prepare samples for analysis using the PacBio RS II system. If any of these protocols are to be used in a production environment, it is the responsibility of the end user to perform the required validation. Guidelines for Using the Sage Science Pippin Pulse Overview Pulsed-field gel electrophoresis is a strategy for resolving large fragments of DNA for analysis. When running a typical direct-current agarose gel, fragments above kb can migrate in a non-predictable manner (not related to their size). Pulsed-field gels work by shuttling DNA back and forth in the gel, effectively slowing down large DNA fragments that might otherwise run at the same rate as smaller ones. By switching the direction of the electric field in a gel, DNA will change its direction of migration. Since smaller molecules can change direction faster than larger molecules, more differentiated separation can be achieved by rapidly switching, or pulsing, the electric field. A single-pulse duration typically separates a relatively narrow range of DNA sizes. For best results, it is necessary to use a range of pulse times; this is accomplished by using a ramp, progressively increasing the forward and/or reverse intervals from a lower limit to an upper limit. The Pippin Pulse is programmed by entering a forward time step and reverse time step. A ramp is programmed by adding a time increment to either or both steps. An accelerated ramp can be achieved by adding additional time increments to either or both steps. Lastly, the number of time steps is also entered, and the steps will continuously cycle for the duration of the run. Cycling allows the same protocol to be run for different lengths of time reproducibly. Page 1

2 NOTE: If the reverse time is not long enough, large molecules may not completely change direction during the reversal, and may actually migrate faster than shorter molecules. This effect can be overcome by using a time ramp where the longest reverse time is long enough to separate all molecules of interest. Page 2

3 Materials Needed Item Vendor Part Number Electrophoresis Reagents 10X KBB Electrophoresis Buffer SeaKem Gold Agarose DNA Markers 2.5 kb Molecular Ruler CHEF DNA Size Standard 8-48 kb Staining/Imaging FlashGel 5X Loading Dye 1X Elution Buffer SYBR Safe DNA Gel Stain Equipment Pippin Pulse Power Supply 15 cm X 15 cm Gel Tray Sub-Cell GT Cell System Kimble 500 ml Bottle KS 260 Basic Shaker Alpha Imager HP Sage Science Lonza BioRad BioRad Lonza Pacific Biosciences Life Technologies Sage Science Bio-Rad Bio-Rad Cole-Parmer IKA Protein Simple KB S33102 PP EW Preparing and Running the Gel The following procedure is for 15 cm x 15 gel tray. The required volume of melted agarose depends on the size of the gel tray. 1. Prepare 3 liters of 0.5X KBB Electrophoresis Buffer. a. In a 1 Liter graduated cylinder, add 50 ml of 10X KBB Electrophoresis Buffer to 950 ml of deionized water. Repeat 2 more times for a total of 3 Liters. 2. Prepare a 0.75% Agarose Gel. a. On an analytical balance, carefully measure out g of Lonza SeaKem Gold Agarose. b. Aliquot 150 ml of 0.5X KBB Electrophoresis Buffer into a 500 ml Kimble bottle. c. Pour the g of Lonza SeaKem Gold Agarose to the 150 ml of 0.5X KBB Electrophoresis Buffer. Mix the solution while adding the agarose to prevent clumping. d. Place bottle in a microwave and heat on high for 1 2 minutes or until powder is fully dissolved. Caution: Bottle and content will be hot. Handle with care. a. Set the IKA Shaker to 50 rpm. Place the bottle on the shaker and shake gently for 10 minutes for solution to cool off. Note: After 10 minutes of shaking, the bottle should be cool enough to touch. However, further cooling may be required. Do not let the solution cool too long because it will solidify in the bottle. b. Pour the solution into the gel tray with the 20-well comb in place. Note: Pour slowly to avoid bubbles which will disrupt the gel. Bubbles can be managed by pushing them toward the edges with a pipette tip. c. Let the gel sit at room temperature for 30 minutes until the gel is fully solidified. After 30 minutes, it can be stored at 4 C until ready for use. d. When ready to use, place gel tray into gel box (see figure 4). Page 3

4 Fig 4. The Bio-Rad Sub-Cell GT Agarose Gel Electrophoresis System 3. Prepare samples. a. Prepare up to 18 samples at 100 ng per well. If the required quantity is not available, 60 ng of DNA may also work. Component Volume Sample X µl of 100 ng Lonza FlashGel 5X Loading Dye 4 µl 1X Elution Buffer Add to bring up to total volume Total 18 µl 4. Prepare DNA Markers. We highly recommend using the following gel markers loaded on adjacent wells. Bio-Rad 2.5 kb Molecular Ruler a. Add 1 µl Bio-Rad 2.5 kb Molecular Ruler + 5 µll Lonza FlashGel 5X Loading Dye + 12 µl 1X Elution Buffer for a total of 18 µl. CHEF DNA Size Standard 8-48 kb a. Prepare a 1:4 dilution of the CHEF DNA Size Standard. Add 1 µl of the CHEF DNA Size Standard + 3 µl 1X Elution Buffer. b. Add 1 µl of the diluted CHEF DNA Size Standard + 5 µl Lonza FlashGel 5X Loading Dye + 12 µl 1X Elution Buffer for a total of 18 µl. Fig 5. Bio-Rad DNA Markers. A. 2.5 kb Molecular Ruler consists of 14 bands in 2.5 kb increments from 2.5 kb to 35 kb. B. CHEF DNA Size Standard 8-48 kb consists of 13 bands: 8.3 kb to 48.5 kb. Page 4

5 5. Run the Pippin Pulse. a. Submerge the gel beneath 6 mm of 0.5X KBB Electrophoresis Buffer in the gel box. b. Load DNA samples and markers onto gel (18 µl per well). c. Connect the electrodes to the Pippin Pulse. d. Turn the Pippin Pulse on by pressing the power button on the front panel. e. Launch the Pippin Pulse v1.32 application by double-clicking the Pippin Pulse icon. f. In the Waveform Type dialog box, select the 10-48kb protocol (the selected protocol will be highlighted in blue). g. For the target duration, enter 16:00 hours. Note that the default value is 2 hours. h. Click START. Fig 6. Pippin Pulse v1.32 Software 6. Staining the gel. a. Make a 1X concentration of SYBR Safe DNA Gel Stain: 30 µl SYBR Safe DNA Gel Stain ml 0.5X KBB Electrophoresis Buffer. b. Place the gel into a 4.5 L Pyrex dish (400 mm x 270 mm) and pour the staining solution over the gel ensuring at least a half centimeter depth of coverage. c. Cover the Pyrex dish with aluminum foil to prevent dye degradation. d. Shake the gel and stain gently for 1 hour. Place the covered Pyrex dish on an IKA shaker and set shaker to 50 rpm. 7. Imaging the gel. a. Image gel according to manufacturer s user guide. Page 5

6 Fig 7. Examples of sheared gdna run on FIGE using a Pippin Pulse. 100 ng of sample was loaded per well and run for 16 hrs. The gel was imaged using the Alpha Imager HP system. For Research Use Only. Not for use in diagnostic procedures. Copyright 2015, Pacific Biosciences of California, Inc. All rights reserved. Information in this document is subject to change without notice. Pacific Biosciences assumes no responsibility for any errors or omissions in this document. Certain notices, terms, conditions and/o r use restrictions may pertain to your use of Pacific Biosciences products and/or third p arty products. Please refer to the applicable Pacific Biosciences Terms and Conditions of S ale and to the applicable license terms at nses.html. Pacific Biosciences, the Pacific Biosciences logo, PacBio, S MRT, SMRTbell and Iso-Seq are trademarks of Pacific Biosciences. BluePippin and SageELF are trademarks of Sage Science, Inc. NGS-go and NGSengine are trademarks of GenDx. All other trademarks are the sole property of their respective owners. Page 6