### **Capillary Electrophoresis Process**

###  **SOP: Production Capillary Electrophoresis on the MegaBACE 1000**

> Version Number: 4
>
> Start Production Date: 3/02
>
> Author: Lisa Kegg/Metzalli Guel
>
> Reviewed by: Lyle Probst
>
> SOP Change Document:

# Summary

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

# Materials & Reagents

> **[Materials/Reagents/Equipment]{.underline} [Vendor]{.underline}
> [Stock Number]{.underline}**

##### [Disposables]{.underline}

> 96 well cycle plate Robbins Scientific 1055-90-0
>
> 96 well cycle plate DOT-Sorenson 96MGB-50
>
> Parafilm "M" American National Cam
>
> Clear Plate Sealers Edge BioSystems 48461

##### [Stock Solutions]{.underline}

> Loading Buffer Amersham US79676
>
> Labeled DNA samples JGI -- RCA Group

##### [Reagents]{.underline}

> Matrix (LPA)/Loading Buffer Amersham US79676
>
> Milli-Q water Milli-Q

##### [Equipment]{.underline}

> MegaBACE 1000 Molecular Dynamics
>
> 96 Plate Washer Tecan
>
> Plate Vortexer VWR
>
> Centrifuge 5417 Eppendorf
>
> Centrifuge 5416 Eppendorf
>
> 96 well Heating Blocks VWR
>
> Ice Bucket VWR

# Procedure

**[Prerun Set-up]{.underline}**

**[Matrix (LPA) Preparation]{.underline}**

1.  Place the Matrix (LPA) tubes in the gray PVC blocks designed for the
    Matrix tubes. If any matrix tubes are leaking or empty place them in
    the box marked "bad matrix". These can be sent back to Amersham.
    Four blocks will fit into the centrifuge at one time.

2.  Centrifuge the Matrix (LPA) at 3000 rpm for 2 minutes. Check the
    weight of the gray bases (written on the side of each) in order to
    balance them out in the centrifuge.

3.  Uncap the Matrix. The uncapping of Matrix should be divided evenly
    between all employees in the capillary group. Each person should
    spread out the uncapping of their matrix through out their shift.
    Refer to breakdown of shift expectations posted in each lab.

4.  Seal with a plate sealer.

5.  Label with lot number and date uncapped.

6.  Place in 4 C refrigerator until use.

**[Sample Assigning and Set-up]{.underline}**

1.  Note which MegaBACE's are down.

2.  Select samples from the main sample freezer in Room 122. Priority
    samples should be selected first then production samples. Check the
    date and batch number and select the oldest production samples
    first.

3.  Assign a MegaBACE number to each sample and write it on the white
    part of the seal with a felt tip pen. Forward and Reverses paired
    plates should be assigned to the same MegaBACE on consecutive runs.

4.  Place the samples back in the --20 freezer until they need to be
    prepared prior to loading the MegaBACE's

**[Preparation of DNA Samples]{.underline}**

1.  Samples can be prepared up to 30 minutes prior to a run.

2.  Remove the samples from the black bases.

3.  If the samples are frozen, place them on the VWR 96-well heating
    blocks for 30 seconds at a temperature of 90 C.

4.  Place up to 8 sequencing plates containing 10 uL of loading solution
    on the VWR Vortexer using the orange rubber separators between the
    samples. Vortex on setting 6 for 2 minutes.

5.  Centrifuge up to 8 plates in the Beckmen at 1000 rpm for 1 minute.

6.  If a plate is dropped, centrifuge again at 1000 rpm for 1 minute.
    Check to make sure the volume is consistent in all wells.

7.  Place samples on the VWR 96-well heating blocks for 2 minutes at a
    temperature of 90 C.

8.  Place the samples on ice and cover with foil until loading.

**[MegaBACE Set-Up and Injection]{.underline}**

1\. Check the sample to see if it has unusual run conditions (if
nothing's indicated on the sample, assume the normal run conditions
apply and go to step 2). If you need to change the run conditions for
any plate, do the following:

a)  On the "Plate Setup" screen of the ICM (Instrument Control Manager),
    select "Configure" from the top menu bar, and then click on "Edit
    mode."

b)  Click on the "New" button near the bottom of the ICM, and type in
    the modified run conditions in the appropriate box(es).

c)  Scan in your sample, and if not automatically saved for you, click
    on the "Save" button near the bottom of the ICM.

2\. Scan the samples into the "Plate Setup" screen of the ICM on the
appropriate MegaBACE and save.

3\. To start the run, go to the ICM and select the Instrument Control
tab.

4\. Select matrix fill and prerun and press start.

5\. The LCD panel on the MegaBACE will direct the operator through the
filling of LPA, pre-running, and injecting sample.

6\. While loading matrix tubes, buffer plates, and water troughs, be
careful not to slam the drawers. If drawers are hard to open/close most
likely it is due to dried matrix and the drawer will need to be pulled
out and wiped down with Milli-Q water by an instrumentation person.

7\. Clean the anode and cathode stages by wiping with a damp towel as
needed. Keeping the stages clean allows for smooth drawer movement and
can prevent capillaries from sticking to the plates/stages/electrodes
and breaking or bending.

8\. During the pre-run time the water troughs should be emptied, rinsed
and filled with fresh Milli-Q water on each run.

9.  Electrophoresis will run for 160 minutes at 7kV. Bring up the "*Run
    Image*" screen on each MegaBACE. Also check the current monitor by
    going to the "*Options*" pull down menu and clicking on "*Current
    Monitor.*" The current should be between 4 and 6 kV.

10. After you have completed the loading process, scan your sample
    plates into the database in the *Run scan in for MegaBACE 1000* to
    ensure that there will be a record of your running those samples.

11. Use the main computer in Rm. 122 and scan your plates into the Excel
    "Run log."

12. Store your sample plates in the appropriate freezer (according to
    the room in which you loaded the samples) for a minimum of 5 days.

> **[MegaBACE Problems]{.underline}**

1\. If a machine experiences problems, report it to your Team Lead and
change the MegaBACE status in the database when you scan in the plates.
Also, write on the MegaBACE itself, using the "Notepad" icon on the
desktop, making sure to include the following information: date, time,
brief description of problem, and your initials.

2.  If a machine goes down, store capillaries (SOP: Store Capillaries
    Protocol for MegaBACE 1000) in water on both anode and cathode
    sides. If a plate should get stuck or any time the anode or cathode
    is stuck in the down position, immediately notify a MegaBACE
    instrumentation member so that they remove the arrays before the
    matrix dries up. This will prevent clogged caps and allow us to
    continue the use of the arrays.

3.  Before bringing a machine back into production, make sure to check
    text messages on the computer screen to insure that the
    instrumentation person is finished working on it.

**[Instrument Parameters]{.underline}**

Matrix Fill/High Pressure Time: 200 sec Matrix Flush Time 1: 22 sec

Matrix Fill/Relaxation: 5 min Matrix Flush Time 2: 7 sec

Prerun Time: 5 min Low Pressure Time: 240 sec

Prerun Voltage: 10kV User Input Time: 100 sec

Preinjection Voltage: 10kV Preinjection Time: 15 sec

PMT1 Voltage: 750 V PMT 2 Voltage: 750 V

Run Temperature: 44 C

**[Injection Conditions]{.underline}**

Sample Injection Voltage: 1.6kV Sample Injection Time: 36 seconds

Run Voltage: 7kV Run Time: 160 minutes

**[Data Analysis]{.underline}**

After completion of the run, a program will execute. This program
automatically initiates base calling with Cimmaron 2.19.05 and then
exports to .abd files. Once 96 files have been called, the program will
rename the folder names, make contact with the FTP server and send all
files. Once these are sent the raw and analyzed data files are moved
from their folders and placed in the d: drive of the MegaBACE's computer
in a sent --raw/processed folder.

# Reagent/Stock Preparation

**[Buffer Preparation]{.underline} (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 is less than 100mL, adjust by adding additional buffer from
    the same lot number. If volume is greater than 100mL remove enough
    buffer to equal 100mL.

2.  Q.S. to 1000mL with Milli-Q water.

3.  Cover the graduated cylinder with parafilm and gently invert 5 times
    to mix completely.

4.  Fill bottles and store in refrigerator when not in use. Label
    bottles with lot number, date, time and initials of preparer.

5.  Prime the Tecan 96PW with buffer until buffer is being dispensed
    from all 96 tips.

6.  Change to the dispense setting on the Tecan 96PW.

7.  Check that the Tecan 96PW is set to dispense 200uL.

8.  Dispense loading buffer into a 96-well plate.

9.  Check that all 96 wells are being filled.

10. Seal 96 well plate with a clear plate sealer.

11. Repeat steps 10,11, and 12 as necessary.

12. Centrifuge at 1000 rpm for 1 minute to remove bubbles.

13. Sealed plates are stored in a 4 C fridge until ready for use.

14. When finished with the Tecan 96PW prime (2-ten second cycles) and
    store with Milli-Q water.

15. If the Tecan PW is not working correctly submit a work request.