technology: an introduction to ga 310 instrument and troubleshooting
DESCRIPTION
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. - PowerPoint PPT PresentationTRANSCRIPT
Technology:An Introduction to GA 310 Instrument
and troubleshooting
Scanner
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)