technology: an introduction to ga 310 instrument and troubleshooting

Technology:An Introduction to GA 310 Instrument

and troubleshooting

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Plates with acrylamide gel

Laser

CCD Camera

Instruments overview Detection on 377

Capillary/ies

CCD Camera

Buffer

Laser

Instruments overview Detection on 310

ABI Prism Technology From filter wheel to CCD camera

PMT

CCD Camera

Laser

370 373

377 310 31003730

“gel” flow

DNA migration

Capillary electrophoresisElectroendosmotic flow

silica

GS entangled polymer

Capillary electrophoresisDynamic coating of capillary

Instruments 377 / Acrylamide gel

Instruments 310 / Capillary electrophoresis

Instruments 310 / Capillary electrophoresis

Includes the positioning mechanism and the carrier which accommodates 48 or 96 tube trays

Instruments 310 / Autosampler tray

> syringe

> syringe drive

> the pump block

Instruments 310 / Pump block

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 electrophoresisElectrokinetic injection

Instruments overview CCD camera detector

512 pixels

VIRTUAL FILTER

Dump

Read

64

100%

75%

25%

0%

Sequencing s/w overview Spectral overlap of dyes

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 dilution Spin column Gel purification

Exo I / SAP treatment Ammonium 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: OD260 / OD280

• Fluorometer

Sequencing overview Template amount evaluation

25 7550 100

X

ladder

Y

Seq. troubleshooting Too much DNA

TOP HEAVYNOT USABLE

signal too strong

NOT USABLE signal too

weak

TOP HEAVYNOT USABLE signal too

strong

NOT USABLEsignal too

weak

OK

Seq. troubleshooting Too much DNA

Seq. troubleshooting Too strong signal

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.

free dye blobs

Seq. troubleshooting Dye terminators contamination

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

Seq. troubleshooting Free dye terminators removal

obsole

te

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 ddH2O).

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).

EtOH 70% cleaning

Instr. related problems Capillary window cleaning (310)