Summary

To perform automated high throughput capillary electrophoresis using the MegaBACE 1000 Sequencing Instrument.

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.