Typing experiments with autofluorescence
Typing experiments with autofluorescence
The typing methods described in this unit are needed when there are a large number of samples with multiple microsatellite markers to be typed. For example, a disease linkage analysis study using a whole gene seizure approach would require 300 markers in 500 individuals, which would require 150,000 genotyping passes. The techniques described in this unit were performed using Perkin-Elmer's automated DAN sequencing system, but can be modified for use with other automated fluorescent sequencing systems.
Operation method
PCR amplification of SSLP for automated fluorescent typing experiments
Materials and Instruments
DNA Sample Move 2. Remove the clip from the 6$ cmweU-to-read acrylamide glue, rinse off the excess glue with water, and flick the dust off the glass plate. Slowly remove the comb and rinse off the excess acrylamide glue with water. Air dry the glass panels or bake dry the panels in a clean Kimwipe. For a 96-well plate to be sampled, the procedure described below uses three 32-well limbs; two 48-well combs can be used as well. 3. Open the door to the chamber at the front of the automated sequencer and fit the low buffer tank under the chamber. Place a clean gel in the chamber so that its flat surface is exactly opposite the laser chamber, with the bottom of the gel plate on the opposite side of the low tank. Make sure the gum is oriented correctly, with the lugged side inward. 4. Look at the area where the glue was scanned for any pieces of glue stuck to the board, and if necessary, wipe it clean with a damp piece of Kim_wipe. Use the black spacer to lock the glue in the correct position and close the hatch. 5. Adjust the photomultiplier tube (PMT) voltage using the 373A key area, select MainMenu, select Calibration, then select Configure to verify the parameters of the run. Check the default parameters and modify them if necessary. Run time: 5 h30 min1070V Operation will not be terminated in the event of a data communication error. Filter wheel B Laser power is 40 mA/30 mW 6. Mouse and Keyboard Select Scan and Map in the menu window of the Genescan672 collection software, and on the 373A main menu use the main key area to select StartPre-run and then PlateCheck. select FullScan to begin scanning the gel plate. If the read area is clean, the scan will look like a flat line and the map will be all gray on the broad side of the gel. If the scan has distinct peaks and colored bees on the plot, there is crumbled glue or dust in the area of the glass plate where the laser reads. At this point the electrophoresis chamber must be opened and both sides of the glass plate wiped clean. It is best to wipe horizontally when wiping with a damp Kim-a-wipe. Re-check that the adhesive is clean as described above, and if necessary repeat the wiping until the area of the adhesive readout is clean. Scans that show a peak that includes 4 colors are due to irregularities in the gel, which cannot be wiped clean. However, as the buffer runs through the gel this problem will automatically disappear ^ If such a strange peak is encountered, note the X-axis of the peak in the scanning window, this value is equivalent to the channel number and can be used to determine which of the artifact peaks will be displayed in two ^ The channel value determines the number of passes in the 36-well comb, if the gel is irregular then skip this pass during the upsampling. 7. Use the mouse to indicate the lowest color row of the scan, with the coordinates of the 1 and ^ axes in the lower left corner of the scan. Check the PMT value for the bottom row, 3^ axis. The y value should be within 20 units of 800, and the 3; value is adjusted according to the value of the PMT in the Setup menu in the 373A key area. The setup menu is accessed through Caiibration in the main menu. Raising the PMT value also raises the ^ value. 8. Repeat steps 2 to 7 until each gel plate is set up on the electrophoresis chamber with a clean scanning area and the correct PMT value. 9. Secure the upper buffer tank to the safety glass plate by rotating the screw clamp until the washer is flat and hand tightening the clamp. Add IXTBE electrophoresis buffer to the upper buffer tank, being careful not to drip any buffer onto the reading area or wipe it off again and rescan. Fill the upper buffer tank with IXTBE buffer until it is about 0.5 cm above the highest hole and about lcm from the top chamber, fill the lower buffer tank until it is about 1 cm from the top of the tank. make sure that the wires in the lower chamber are covered, and check that the gaskets in the upper buffer tank are properly sealed. Each instrument requires approximately I.5L of 1 X TBE. 10. Using a 60 ml syringe, flush the gel wells with 1 x TBE. Place the top of the needle right between the two glass plates in the buffer tank above. 11. Mango need, place a plastic indicator tape on the outside of the glass plate so that the sequence of the corresponding glue holes can be displayed. Check the glue for broken hole edges, air bubbles, or bad glue holes. Note that if any of the glue holes have these problems they cannot be sampled. 12. Place the plate to be sampled in an ice box and remove the plastic seals from the first 4 columns (A1~A4). Set the micropipette to know 1, take up the sample in the hole Al, put the flat surface of the tip between two glass plates, about -half into the first glue hole, slowly pump the sample into the glue hole, pull out the tip. be careful not to overfill the glue hole, to prevent the sample from flowing into the next glue hole. If an adjacent glue hole is contaminated, skip it and mark it. 13. Continue to draw samples from the upper plate up to hole Hl, then move to the next column from hole A2 to H2 solitaire. Place the first four columns of the plate onto the first three glues, and if a wrong glue is loaded, note the wrong lane on the glue hole. 14. When all the gels have been loaded, remove the plastic indicator tape from the glass plate, plug in the top and bottom buffer tanks, and close the door to the electrophoresis chamber. In the sequencing key area of the 373A, press the ChooseRim button, select GenescanRun to start electrophoresis, and immediately use the mouse to indicate the green Coliect button to begin Genescan collection.... The Collect button will begin blinking to verify that collection has begun, if the green button does not begin blinking, press the StopRun button in the 373A key area and get help from someone with experience. 15. Repeat steps 9~14 to upsample A4~H8 and A9~H12 onto the other two gels. When all three instruments have been upsampled and Genescan collection has begun, finally check that the Collect button is blinking. When the gel run is complete, check for errors. The Genescan should not have any errors during collection, and the 373 key area should show that the execution time took 5.5 h. The Genescan should not have any errors during collection. 16. Turn off the 373A sequencer, open the door of the electrophoresis chamber, hold the adhesive plate, with the upper buffer tank still on top, still facing the laser chamber, open the pins on both sides and tear off the black tape from the adhesive丄. The first thing you need to do is to take a look at the picture and see if you can see the picture. 17. Carefully take the lower buffer tank out of the electrophoresis chamber and pour off the buffer, rinse both tanks and put them under the sequencer. Wipe up any spilled liquid in the electrophoresis chamber with a damp paper towel, close the door of the electrophoresis chamber, and repeat this step for the other gel plates. 18. Analyze the strand. Use Genescan Analysis and Genotyper software to analyze the alleles according to the manufacturer's instructions. For more product details, please visit Aladdin Scientific website.
4dNTP mix MgCl2 Fluorescent dye labeled forward primer for each microsatellite marker Forward primer for each microsatellite marker Taq DNA polymerase
96-well plates Oven-drying oven Multi-channel drain pipettes and sterilized reagent trays 96-well plastic sealing film 5 ml and 50 ml tubes Thermocycler
1. Turn on the sequencer and computer.