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

3. Scanner Plates with acrylamide gel Laser CCD Camera Instruments overview Detection on 377

4. Capillary/ies CCD Camera Buffer Laser Instruments overview Detection on 310

5. ABI Prism Technology From filter wheel to CCD camera PMT CCD Camera Laser 370 373 377 310 3100 3 730

6. “gel” flow DNA migration Capillary electrophoresis Electroendosmotic flow

7. silica GS entangled polymer Capillary electrophoresis Dynamic coating of capillary

8. Instruments 377 / Acrylamide gel

9. Instruments 310 / Capillary electrophoresis

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

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

13. 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 Capillary electrophoresis Electrokinetic injection

14. Instruments overview CCD camera detector 512 pixels VIRTUAL FILTER Dump Read 64

15. 100% 75% 25% 0% Sequencing s/w overview Spectral overlap of dyes

16. 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. Sequencing s/w overview What is a matrix / spectral calib.

17. Direct dilutionSpin columnGel purification Exo I / SAP treatmentAmmonium Acetate ppt PCR PRODUCT SEQUENCING REACTIONS Reaction overview Cleaning PCR product

18. 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: OD 260 / OD 280 Fluorometer Sequencing overview Template amount evaluation 257550100 X ladder Y

19. Seq. troubleshooting Too much DNA TOP HEAVY NOT USABLE signal too strong NOT USABLE signal too weak

20. TOP HEAVY NOT USABLE signal too strong NOT USABLE signal too weak OK Seq. troubleshooting Too much DNA

21. Seq. troubleshooting Too strong signal

23. raw data Seq. troubleshooting Too little DNA

24. Actual primer Tm 43.5°C, estimated > 52°C Seq. troubleshooting Noise due to weak signal 42°C 45°C

25. <100 ng plasmid Seq. troubleshooting Noise due to weak signal 300 ng plasmid

26. Seq. troubleshooting Noise due to insertion / deletion Rev. Forw.

27. free dye blobs Seq. troubleshooting Dye terminators contamination

28. Centrisep Columns (PE Biosystems) Multiscreen plate (Millipore) DyeEx or DyeEx96 (Qiagen) EtOH + 3M NaOAc precipitation SAP (BAP) digestion (usb Corporation) Phenol:Chl extraction EtOH (+ MgCl 2 ) precipitation Seq. troubleshooting Free dye terminators removal o b s o l e t e

29. Seq. troubleshooting Degradation of stored seq. product

30. Seq. troubleshooting Matrix (spectral cal.) problem

31. 100%T 45%C Seq. troubleshooting Matrix (spectral cal.) problem

32. Instr. related problems Spike due to CCD current (310) raw data anal. data

33. Polymer related problems Spikes due to old POP or dust raw data anal. data

34. In this case clean properly the capillary window with 70% EtOH and check the water quality (should be ddH 2 O). Unfiltered buffer solution in the buffer vial can generate a lot of spikes in the electropherograms. Instr. related problems High baseline + spikes on 310

35. Capillary related problems Loss of resolution change capillary!!!

36. Instr. related problems Syringe 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).

37. EtOH 70% cleaning Instr. related problems Capillary window cleaning (310)