Capillary Electrophoresis |
Version Number: | 1 |
Start Production Date: | 6/9/00 |
Author: | Susan Lucas |
Edited by: | X |
Reviewed by: | Heliodoro Cardenas |
Summary |
Materials & Reagents |
Materials/Reagents/Equipment | Vendor | Stock Number |
---|---|---|
Disposables | ||
96 well cycle plate | Robbins Scientific | 1055-90-0 |
Parafilm "M" | American National Cam | N/A |
Clear Plate Sealers | Edge BioSystems | 48461 |
Stock Solutions | ||
Loading Buffer | JGI | See Below |
Labeled DNA samples | JGI | From Seq. Chem. |
Reagents | ||
LPA/Loading Buffer | Amersham | US79676 |
Milli-Q water | Milli-Q | N/A |
Equipment | ||
MegaBACE 1000 | Molecular Dynamics | N/A |
Multidrop 96 well | Titertek | N/A |
Plate Vortexer | VWR | N/A |
500 mL Glass Bottle | VWR | N/A |
Centrifuge Allegra 6R | Beckmen | N/A |
Centrifuge 5417 | Eppendorf | N/A |
96 well Heating Blocks | VWR | N/A |
Ice Bucket | VWR | N/A |
Procedure |
Preparation of DNA Samples
1. Place 6 sequencing plates containing 10 uL of loading solution on the VWR Vortexer and vortex on setting 6 for 2 minutes.
2. Centrifuge plates in the Beckmen at 1000 rpm for 1 minute.
3. Place on the VWR 96-well heating blocks for 2 minutes for a temperature of 90 C.
4. Leave on ice until loading.
Preparation of MegaBACE and Injection
1. Prepare a sample sheet for the DNA sample that you will be loading using the sample sheet generator on Netscape. Be sure to include the correct machine number, library, plate number and direction of primer.
2. Select the Standard run condition template in the ICM and Save the setup.
3. Centrifuge the LPA at 3000 rpm for 2 minutes.
4. Centrifuge the buffer plates at 1000 rpm for 1 minute. Check for bubbles. If present then spin again.
5. Follow the Standard run condition template for filling the MegaBACE with LPA, Prerunning, and injecting sample. The Run condition Template can be set up in the template file located in the c:/Program Files/Molecular Dynamics/MegaBACE/Templates using the information provided below.
6. Electrophoresis will run for 120 minutes at 8kV.
Run Parmeters/Conditons
Run Parmeters/Conditons
Matrix Fill/High Pressure Time: 200 sec | Matrix Flush Time 1: 22 sec |
Matrix Fill/Relaxation: 10 min | Matrix Flush Time 2: 7 sec |
Prerun Time: 5 min | Low Pressure Time: 240 sec |
Prerun Voltage: 10kV | User Input Time: 60 sec |
Preinjection Voltage: 10kV | Preinjection Time: 15 sec |
PMT1 Voltage: 750 V | PMT 2 Voltage: 750 V |
Run Temperature: 44 C |
Run Condition Template
Sample Injection Voltage: 2kV | Sample Injection Time: 75 seconds |
Run Voltage: 8kV | Run Time: 120 minutes |
Data Analysis
Using the MegaBACE 1000 Sequence Analyzer (version 2), data is processed with the Cimarron Basecaller. Files are then converted to abd and transferred to unix for PHRED analysis.
Reagent/Stock Preparation |
Buffer Preparation (1X)
1. Measure out 2 50mL bottles of Running Buffer of the same lot number in a 1000mL graduated cylinder. Final volume should be 100mL. If volume does not measure 100mL, adjust by adding additional buffer from the same lot number.
2. Q.S. to 1000mL with Milli-Q water.
3. Cover the graduated cylinder with parafilm and invert 5 times.
4. Fill 2 500 mL VWR bottles and store in fridge when not in use. Label bottles with lot number of buffer.
5. Check settings on multidrop. Column = 12, Volume = 200 uL
6. Prime the multidrop with buffer until liquid is being dispensed from all 8 tips.
7. Using the multidrop, dispense loading buffer into a 96-well cycle plate.
8. Seal 96 well plate with a clear plate seal and centrifuge to remove bubbles.
9. Sealed plates are stored in a 4 C fridge until ready for use.