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
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.
Direct dilutionSpin columnGel purification Exo I / SAP treatmentAmmonium Acetate ppt PCR PRODUCT SEQUENCING REACTIONS Reaction overview Cleaning PCR product
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 X ladder Y
Seq. troubleshooting Too much DNA TOP HEAVY NOT USABLE signal too strong NOT USABLE signal too weak
TOP HEAVY NOT USABLE signal too strong NOT USABLE signal too weak OK Seq. troubleshooting Too much DNA
Seq. troubleshooting Too strong signal
raw data Seq. troubleshooting Too little DNA
Actual primer Tm 43.5°C, estimated > 52°C Seq. troubleshooting Noise due to weak signal 42°C 45°C
<100 ng plasmid Seq. troubleshooting Noise due to weak signal 300 ng plasmid
Seq. troubleshooting Noise due to insertion / deletion Rev. Forw.
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
Seq. troubleshooting Degradation of stored seq. product
Seq. troubleshooting Matrix (spectral cal.) problem
100%T 45%C Seq. troubleshooting Matrix (spectral cal.) problem
Instr. related problems Spike due to CCD current (310) raw data anal. data
Polymer related problems Spikes due to old POP or dust raw data anal. data
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
Capillary related problems Loss of resolution change capillary!!!
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).