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Technology: An Introduction to GA 310 Instrument and troubleshooting

Technology: An Introduction to GA 310 Instrument and troubleshooting. CCD Camera. Laser. Scanner. Plates with acrylamide gel. Instruments overview Detection on 377. Capillary/ies. Laser. CCD Camera. Buffer. Instruments overview Detection on 310.

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Technology: An Introduction to GA 310 Instrument and troubleshooting

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  1. Technology:An Introduction to GA 310 Instrument and troubleshooting

  2. CCD Camera Laser Scanner Plates with acrylamide gel Instruments overviewDetection on 377

  3. Capillary/ies Laser CCD Camera Buffer Instruments overviewDetection on 310

  4. ABI Prism TechnologyFrom filter wheel to CCD camera 370 373 PMT Laser 377 310 3100 3730 CCD Camera

  5. Capillary electrophoresisElectroendosmotic flow DNA migration “gel” flow

  6. Capillary electrophoresisDynamic coating of capillary GS entangled polymer silica

  7. Instruments 377 / Acrylamide gel

  8. Instruments 310 / Capillary electrophoresis

  9. Instruments 310 / Capillary electrophoresis

  10. Instruments 310 / Autosampler tray Includes the positioning mechanism and the carrier which accommodates 48 or 96 tube trays

  11. Instruments 310 / Pump block > syringe > syringe drive > the pump block

  12. Capillary electrophoresis Electrokinetic injection Capillary and electrode are placed into the sample Voltage is applied “-” charged DNA enters the capillary as it migrates toward the “+” electrode at the other end of the capillary

  13. Read Dump Instruments overviewCCD camera detector 64 512 pixels VIRTUAL FILTER

  14. 100% 75% 25% 0% Sequencing s/w overviewSpectral overlap of dyes

  15. Sequencing s/w overviewWhat is a matrix / spectral calib. The matrix is used to filter raw data in order to “extract” the rigt value for each color. Raw data still has signals also in the “wrong” colors, the multicomponent analysis subtracts the values for each peak giving a clear result.

  16. Reaction overviewCleaning PCR product PCR PRODUCT Direct dilution Spin column Gel purification Exo I / SAP treatment Ammonium Acetate ppt SEQUENCING REACTIONS

  17. X Y ladder 25 50 75 100 Sequencing overviewTemplate amount evaluation It is very important to roughly estimate the amount of template DNA • Agarose gel (0.7 to 1.2 %) stained with EtBr + ladder or, even better, a scalar amount of a well quantified template (i.e. linearized pGEM) • Spectrometer: OD260 / OD280 • Fluorometer

  18. NOT USABLE signal too strong NOT USABLE signal too weak TOP HEAVY Seq. troubleshootingToo much DNA

  19. TOP HEAVY NOT USABLE signal too strong NOT USABLE signal too weak OK Seq. troubleshootingToo much DNA

  20. Seq. troubleshootingToo strong signal

  21. Seq. troubleshootingToo strong signal

  22. Seq. troubleshootingToo little DNA raw data

  23. 45°C 42°C Seq. troubleshootingNoise due to weak signal Actual primer Tm 43.5°C, estimated > 52°C

  24. Seq. troubleshootingNoise due to weak signal <100 ng plasmid 300 ng plasmid

  25. Seq. troubleshootingNoise due to insertion / deletion Forw. Rev.

  26. Seq. troubleshootingDye terminators contamination free dye blobs

  27. Seq. troubleshootingFree dye terminators removal Centrisep Columns (PE Biosystems) Multiscreen plate (Millipore) DyeEx or DyeEx96 (Qiagen) EtOH + 3M NaOAc precipitation SAP (BAP) digestion (usb Corporation) Phenol:Chl extraction EtOH (+ MgCl2) precipitation obsolete

  28. Seq. troubleshootingDegradation of stored seq. product

  29. Seq. troubleshootingMatrix (spectral cal.) problem

  30. T 100% C 45% Seq. troubleshootingMatrix (spectral cal.) problem

  31. Instr. related problemsSpike due to CCD current (310) raw data anal. data

  32. raw data anal. data Polymer related problemsSpikes due to old POP or dust

  33. Instr. related problemsHigh baseline + spikes on 310 In this case clean properly the capillary window with 70% EtOH and check the water quality (should be ddH2O). Unfiltered buffer solution in the buffer vial can generate a lot of spikes in the electropherograms.

  34. Capillary related problemsLoss of resolution change capillary!!!

  35. Instr. related problemsSyringe problem (waterfall) Wash new syringes carefully with 60°C HPLC water from Merck, and then good with cold water. Sometimes its necessary and helps to wash new syringes with 2 N NaOH (prepared with HPLC water from Merck and afterwards with cold HPLC water).

  36. EtOH 70% cleaning Instr. related problemsCapillary window cleaning (310)

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