Dr. Bextine DNA Sequencing Beckman Coulter CEQ-8000 1. Sample Amplification using PCR A. 20 Вµl reaction volume is used, no SYBRGRN (traditional PCR). 2. Gel Electrophoresis: The PCR product needs to be run through gel electrophoresis (5 Вµl of PCR product at approximately 75 volts) in order to check if the PCR successfully amplified the DNA of interest. 1. 5 Вµl of the standard ladder should be run 2. Check to see if the PCR sample band appear at a position corresponding to the correct fragment length of the primer thatвЂ™s used. 3. PCR Product Purification: The PCR product must be purified to remove impurities from the sample. This is done through the Qiaquick PCR Purification kit (See Qiaquick manual for protocol p.18). NOTE: Add ethanol (96%-100%) to Buffer PE before use, all centrifuge steps are at 13,000 rpm in a conventional tabletop microcentrifuge. A. Add 5 volumes of Buffer PB to 1 volume of the PCR sample and mix. B. Place a QIAquick spin column in a provided 2mL collection tube. C. To bind DNA, apply the sample to the QIAquick column and centrifuge for 1 minute D. Discard flow-through. Place the QIAquick column back into the same tube. E. To wash, add 750Вµl Buffer PE to the QIAquick column and centrifuge for 1 minute. F. Discard flow-through and place the QIAquick column back in the same tube. Centrifuge for an additional 1 minute. G. Place QIAquick column in a clean 1.5mL microcentrifuge tube. H. To elute DNA, add 50Вµl Buffer EB to the center of QIAquick membrane and centrifuge the column for 1 minute. 4. Sequencing PCR: After the PCR product has been purified and the concentration identified, a second PCR reaction will need to be done. This PCR reaction is unique in that the amount of template DNA used must fall within a certain concentration range, only one primer is used, and it involves ddNTPвЂ™s as wells as dNTPвЂ™s. When a ddNTP is incorporated into a strand which is being synthesized, synthesis stops because the ddNTP does not contain a 3Вґ Hydroxyl. The reagents for this PCR reaction are found in the Genome Lab DTCS-Quick Start Kit. The steps for the sequencing PCR are outlined as follows: A. Find the concentration of DNA to be used in the PCR sequencing reaction 1. The concentration DNA must fall in the range of 10-50 fmol 1 2. By using the following figure, you can determine the number of fmol contained per microliter by the size of the DNA fragment and the amount of DNA in ng per microliter. (For instance, a 0.2 kilobase fragment at a concentration of 3.3 ng/Вµl will be 25 fmol/Вµl. 1 Вµl of DNA should be used in the PCR sequencing reaction since this is equivalent to 25 fmol, which falls in the middle of the acceptable range.) B. Perform the Sequencing PCR (Beckman Coulter protocol states to use 8 Вµl of DTCS Quick Start Mix. However, for cost effectiveness, the Bextine lab will run 1/2 reactions. Therefore, 4 Вµl of DTCS Quick Start Mix will be used in the reaction.) 1. Only one primer should be used in the reaction 2. The amount of primer and DTCS Quick Start Mix used is constant. The amount of DNA in Вµl to be used is determined so that the amount falls within the acceptable 10-50 fmol range. Water is used to adjust the total volume of the reaction to 20 Вµl. 20 ul Total Volume Reaction Reagent Water DNA Primer DTCS Quick Start Mix 2 Amount 12 Вµl 2 Вµl 2 Вµl 4 Вµl 3. The samples should be loaded into the thermocycler and the following protocol can be followed: A. Select F1: Library Protocol B. Scroll Down to CEQ and press Enter C. Select Run Protocol and press Enter D. Adjust the volume to the amount of volume per sample E. Select F5: Begin Run 4. A good stopping point is after the sequencing PCR reaction has finished. The samples can be stored in either the freezer or refrigerator. 5. Ethanol Precipitation: The purpose of this step is to precipitate DNA from the sequencing reaction. The protocol is outlined below: 1. Set the MCT centrifuge to 2В°C 2. Prepare a labeled 0.5 ml MCT for each sample 3. Prepare fresh Stop Solution (per each sample): 2 Вµl of 3M NaOAc (pH 5.2), 2 Вµl of 100 mM NA2-EDTA (pH 8.0), and 1 Вµl of 20 mg/ml of glycogen (The glycogen is supplied with the Genome Lab DTCS-Quick Start Kit.). Since this preparation of Stop Solution is for one sample, the above volumes should be adjusted according to how many samples there are. (For 8 samples prepare 20 Вµl NaOAc, 20 Вµl of NA2-EDTA (pH 8.0), and 10 Вµl glycogen.) Spin down the stop solution after preparation and vortex thoroughly. 4. Add 5 Вµl of the Stop Solution to each MCT. 5. Transfer the Sequencing PCR product to the appropriately labeled MCT. 6. Vortex thoroughly 7. Add 60 Вµl of ice cold 95% ethanol from the -20В°C freezer and vortex thoroughly. 8. Immediately incubate the samples at -81oC for 30 minutes. Then, centrifuge at 14,000 rpm for 20 minutes. 9. Remove the supernatant with a micropipetter being careful not to dislodge the pellet. 10. Wash the pellet by adding 200 Вµl of ice cold 70% ethanol from the -20В°C freezer. The ethanol can be applied to the side of the MCT as care should be taken not to dislodge the pellet. 11. Centrifuge at 14,000 RPM for 7 minutes. 12. Remove the supernatant with a micropipetter being careful not to dislodge the pellet. 13. Repeat steps 10-12 once 14. Dry the pellet so that no ethanol remains in the MCT. Vacuum drying can be done to speed the ethanol evaporation. The vacuum should be turned on and off slowly. 15. Resuspend the sample by adding 40 Вµl of Sample Loading Solution (provided in the Genome Lab DTCS-Quick Start Kit) to the sample in the MCT. 16. Vortex (Note: This can be a good stopping point. The MCTвЂ™s can be stored overnight in the freezer. It is a good idea to cover the MCT with a foil.) 17. Transfer the sample to the appropriate well in the sample plate 3 18. Spin down the plate at approximately 300 RPM for 30 seconds 6. Sample Preparation for Sequencing A. Place one drop of light mineral oil (provided in the Genome Lab DTCS-Quick Start Kit) into each well B. Load the sample plate in the Beckman-Coulter CEQ 8000 7. Sequencing the Samples A. Setting up the sample plate 1. From the CEQ Analysis Software window, click the SETUP icon. 2. Name the cells corresponding to the samples in the sample plate 3. When making changes to the entire plate, be sure to highlight all of the cells 4. Select a method from the drop-down menu at the bottom of the sample set 5. Select the appropriate analysis parameters, as well as report and export parameters if needed. (Select both raw and analyzed data boxes to save the chromatograms as .scf, standard chromatogram files. If you forget to export your data at this time, you can do it later.) 6. Select File/Save As because you must save your work to continue. Saved cells will change color 7. If needed, select the project you created in the database module from the Project Name drop-down menu and click OK B. Replenishing the wetting tray 1. From the CEQ Analysis Software window, click the Run icon 2. Click Replenish/Replace Wetting Tray 3. Open the sample cover when prompted. Remove the wetting tray, empty contents, and fill with distilled water up to the bottom of the letters, NOT TO THE FILL LINE C. Capillary Array (Beckman Coulter recommends that the capillary array should be changed after 60 days, or 100 runs, whichever happens first) 1. Click on Replinish/Release Manifold Plug and the Remove Capillary Array dialog box will appear 2. Remove the manifold plug by opening the sample access cover, then open the capillary access cover by placing hands on the plastic clear cover is latched and pull up 3. Unlatch the two rubber latches holding the temperature control cover and lift it up 4. Loosen the first screw on the manifold access cover and remove the cover 5. Loosen the two screws to the plenum (under the capillary temperature control cover) and remove that cover as well 6. On the right of the machine will be a red eject lever (where the manifold access cover was removed), pull it up to release the manifold plug 7. When you pull out the array plug, hold it in place for 10 seconds so that the acrylamide gel strand solidifies and then remove the manifold plug 4 8. Wipe off all the gel that may be on or around the manifold plug area with a Kimwipe 9. Retrieve the capillary array and remove the plastic coverings 10. Insert the capillary array into the sequencer 11. Put the array fitting cover back on and screw it in 12. Put the capillary cover back on 13. Screw the capillary cover back on and then finally close the capillary access cover and secure the two rubber latches 14. Once the capillary is installed hit OK on the dialog box D. Gel Cartridge Installation 1. Select Replenish/Release Gel Cartridge 2. When the system is ready to release the gel cartridge, the Release Gel Cartridge dialog box will be displayed. Wait until the screw is completely disengaged 3. Open the gel pump access cover by gently pushing on it 4. Pull on the cartridge locking lever (it will pull outward to a 90В° angle) 5. Grasp the wings of the gel plug and pull it outwards, wipe off any gel that may remain on the plug 6. Take cap off gel cartridge (wipe off any gel), place it on plug, then place the plug off to the side 7. Take the gel cartridge and insert it where the plug was 8. Push the cartridge locking lever towards the back of the instrument into its locked position 9. Close the gel access cover 10. In the Remove Gel Cartridge dialog box click on install cartridge, enter in the appropriate information and click Done E. Running the samples 1. Go to Direct Control, click purge manifold 2. Go to Direct Control, click Gel Capillary Fill 3. Go to Direct Control, click Optical Alignment 4. Go to Run, Monitor Baseline, and check enable monitor baseline in the dialog box (If any of the bases are above 6,000, try another optical alignment, clean off the array, or purge the manifold) 5. Then under the Direct Control window, click Load sample plates 6. Load your sample and buffer plate into the appropriate place 7. Then go to Run, start sample plate to begin your run. Make sure that the correct cells are highlighted F. Disassembly of the CEQ-8000 1. Remove the sample and buffer plate from the machine 2. Rinse the wetting tray with distilled water and refill it 3. Remove the gel cartridge, using a Kimwipe to remove the excess gel from the end. Replace the cartridge with the plug. *Revised June 2006. 5