I.
Materials
1.
Provided
by the sequencing facility:
a. Sequencing
kit
1.
Master
mix
2.
Test
primers and plasmid
3.
Sample
loading solution
b. Glycogen
c. Mineral
oil
d. Gel
e. Buffer
f. Buffer
plates
g. Capillaries
2.
Provided
by the user:
a. Reaction
tubes or plates
1.
Recommended
products:
a.
0.2
thin-wall PCR tubes PGC #
b.
Thin-walled
microplates: Costar polypropylene plates Fisher #07200613, Product
Code 6551
b. 100%
molecular biology grade ethanol
c. 3
M Sodium Acetate (pH 5.2)
d. 100
mM Na-EDTA (pH 8.0)
II.
Costs
1.
The
sequencing costs are as follows:
a. Half
reaction - $5.00
1.
The
standard reaction that generally gives an output of 500-700 bp
b. Full
reaction - $8.00
1.
This
reaction may give outputs of 700-800 bp
c. Machine
costs - $2.50
1.
The
cost of running empty wells in a column, for example, if you run 5
reactions on
a plate, the machine costs apply
to
3 wells (to fill the one column of
8).
III.
Sequencing Reaction
1.
Plasmid Reaction Protocol
a. Reactions should be put
together in 0.2
mL thin wall tubes or 96 well microplates. All reagents should be kept on ice during the
preparation.
b. To
the tubes, add the appropriate amount of template DNA (100 ng of
plasmid DNA with
500-1500 bp inserts, for example) and water up to 10 uL.
1.
For all plasmids incubate the DNA at 95C for 1
minute prior to adding to sequence reaction mix.*
a.
This step is very important. It nicks the DNA so that the sequencing reaction
can
proceed. Missing this
step will
drastically reduce the quality of your results. This step is not
necessary for genomic pcr products, only plasmids.
b.
Allow the reaction to cool before proceeding
c. Now in
the following order, add to the DNA:
1.
2uL of 1.6
uM primer
2.
4uL of the
DTCS Quick
Start Master Mix
3.
4ul of 5X Sequencing Buffer - 400mM Tris base
(4.84g/100ml) + 10mM MgCl2 (0.203g/100ml) adjust to ph 9.0 with conc.
HCl. Total volume of reaction should be 20ul.
d. Mix
the components thoroughly and consolidate the reaction at the bottom
of the
tube by shaking or brief centrifugation
2.
Thermal cycling program
a. 30
cycles of:
1.
96 C
20
seconds
2.
50 C
20
seconds
3.
60 C
4
minutes
b. 4 C hold
3.
Ethanol precipitation
a. It
is best to do the precipitation as soon as possible after the thermal
cycling
reaction has completed. Do
not freeze
the samples at this point, but allowing them to sit overnight at 4 C is
acceptable.
b. Prepare
fresh Stop Solution for each precipitation by mixing together 2 uL of 3M
sodium acetate (pH 5.2), 2 uL of 100 mM Na2EDTA (pH
8.0), and
1 uL of 20 mg/mL glycogen for
each
sequencing reaction.
c. Add
5 uL of Stop Solution to each
sequencing reaction and follow the appropriate precipitation protocol
below. Vortex vigorously
for 20-30
seconds.
d. Using
the appropriate protocol below, ethanol precipitate the DNA.
4.
Preparing sample plates
a. Overlay
1 drop of mineral oil to each sample
b. To
any unused wells in a column, add 40 uL
of water, and overlay a drop of mineral oil.
c. Prepare
a buffer plate using the flat-bottomed culture plates. Fill the wells
corresponding to samples in your sample
plate
2/3 –
3/4 full of buffer.
d. After
the reaction, samples can be kept at -20 C and re-run on the machine if
necessary.
*See Beckman recommendations for variations on this plasmid DNA nicking.
IV.
Opening the CEQ8000 Software
1.
Turn
on PC and wait for windows to start
2.
If
not already on, press power switch to ON (Fig.1: #9)
3.
Click
the CEQ8000 icon on the desktop to open the window shown in the
picture
4.
There
are 7 different modules available on this screen, most of which will
be
discussed later.
a. Sample
Setup – used to set up the sample plates
b. Run
– used to interact with the sequencing machines, and actually
running
your sequences
c. Sequence
Analysis – used to look at your results
d. CEQuence
Investigator
e. Fragment
Analysis
f. Databases
– manages where data is stored
g. Exit
– exits the program
II.
Databases
1.
From
the CEQ analysis software menu, select the DATABASE module icon (Highlighted in Figure
2)
2.
If
you do not already
have a
database:
a. In
the data manager module window (Fig. 3), select File / New
Database
b. In
the New Database dialog
box:
1.
Enter
the name for this new database
2.
Select
Set as Working Database and
click OK
c. If
you wish to further subdivide data storage (otherwise, all the data is
stored
in the default project folder), create a new project folder:
1.
Highlight
the desired database where the project will be
2.
Select
File / New
3.
Highlight
the new project and select File / Rename,
enter in the desired name
3.
If
you do have a
database:
a. Select
File / Set or right
click on the database and click on Set
As
Working Database
4.
For
the Set As Working Database
option to
be available, all other CEQ8000 programs, such as the run window, must
be closed.
III.
Setting up the sample plate
1.
From
the CEQ Analysis Software window or the Shortcut Bar (Fig. 4), click
the SETUP
icon (circled in Figure
4).
2.
In
the plate set-up screen that appears, name the cells corresponding to
the
samples in your actual sample plate (Fig.6)
3.
When
making changes to the entire plate, be sure to highlight all of the
cells.
4.
Select
a method from the drop-down menu at the bottom of the sample set.
5.
Select
the appropriate analysis parameters (as well as report and export
parameters if
needed).
a. Select
both raw and analyzed data boxes to save the chromatograms as .scf
(standard
chromatogram files).
b. If
you forget to export your data at this time, you can do it later.
6.
Select
File / Save as, you must save
your work
before you can continue!
Saved
cells will change color.
7.
If
needed, select the project you created in the database module from the
Project
Name drop-down menu and click
OK.
IV.
Preparing the
machine to sequence
1.
Wetting
tray
a. From
the CEQ Analysis Software window or the Shortcut Bar (Fig. 7), click
on the Run
icon
b. Click
Replenish / Replace Wetting Tray
Open the sample cover
(Fig.1: #1)
when prompted by the software. Remove the wetting tray (Fig. 8), empty
any
water in the tray and fill with distilled water up to the bottom of
the
letters, NOT TO THE FILL LINE.
2.
Capillary
array
a. Installation
1.
Click
on Replenish /
Release Manifold
Plug and the Remove Capillary Array dialog box will
appear
2.
Remove
the manifold plug by opening the sample access cover (Fig. 1: #1),
then open
the capillary access cover (Fig. 1: #2) by placing hands where the
plastic clear
cover is latched and pull up
3.
Unlatch
the two rubber latches (Fig. 1: #6) holding the capillary temperature
control
cover (Fig. 1: #5) and lift it up
4.
Loosen
the first screw on the manifold access cover (Fig. 1: #7) and remove
the cover
5.
Loosen
the two screws to the plenum (under the capillary temperature control
cover) and
remove that cover as well
6.
On
the right of the machine will be a red eject lever (where the manifold
access
cover was removed; Fig. 9), pull it up to release the manifold
plug
7.
When
you pull out the array plug, hold it in place for 10 seconds (until
the
acrylamide gel strand solidifies) and then remove the manifold
plug
8.
Make
sure to wipe off all gel that may be on or around the manifold plug
area with a
Kimwipe
9.
Open
the new capillary array and remove all the coverings.
10. Hold the
capillary
and the array by holding the protruding tabs.
11. Place the
capillaries in the wetting tray while inserting the array into the
array
fitting.
12. Then
insert the
capillaries with tab pointing out.
13. Put the
array
fitting cover back on and screw it in.
14. Take your
left
hand and put the capillary cover back on, while hold the capillaries
in the
notch where the cover closes with your right hand.
15. Screw the
capillary cover back on and then finally close the capillary access
cover and
secure the two rubber latches.
16. Once the
capillary
is install hit OK on the
dialog box
17. After
installing a
new capillary array, be sure to purge the manifold (see page 16).
h. Life
span
12. Beckman
recommends
that the capillary array should be changed after 60 days, or 100 runs,
whichever happens first.
i. Monitoring
the baseline
12. To ensure
accurate
analysis of the sequencing runs, monitor the baseline before each run.
13. Click
Run,
monitor baseline. When a dialog appears, check
the box
next to enable monitor baseline.
Or click on the button circled (Figure 10).
14. To view
the
baseline, click on the side tabs, A-H (Figure 11) one at a time. This will show you the
baseline fluorescence
readings. Wait around 30
seconds
to let the readings even out.
a.
There
may be a spike in the baseline at the beginning of a run. If so, drag a square with the
mouse to
zoom in on the part that you are interested in.
15. If any of
the
baselines are greater than 6000 counts, try the following:
a.
An optical alignment
i. At
the direct control screen, click on the optical alignment.
b.
Clean off the array
i. Access
the capillary array as above.
ii. Using
a slightly damp Kimwipe, very gently clean off the capillary
window.
c.
Refill
the capillaries
16. Recheck
the
baselines as above
Gel
Cartridge
a. Installation
1.
Select
Replenish / Release Gel Cartridge
2.
When
the system is ready to release the gel cartridge, the Release Gel
Cartridge dialog box will be displayed. Wait until the lead screw is
completely
disengaged.
3.
Open the gel pump access cover
(next to
the On/off switch) by gently
pushing on
it (it is spring-loaded)
4.
Pull
on the cartridge locking lever (it will pull outward to a 90 degree
angle).
5.
Grasp
the wings of the gel plug and pull it outwards, wipe off any gel that
may
remain on the plug or in the gel cartridge.
6.
Take
cap off gel cartridge (wipe off any gel), place it on plug and place
plug off
to the side.
7.
Take
gel cartridge and insert it where the plug was.
8.
Push
the cartridge locking lever towards the back of the instrument into is
locked
position
9.
Close
the gel cartridge access cover.
10. In the Remove Gel Cartridge
dialog box click on install
cartridge, enter in the appropriate information and
click Done.
4.
Running
the samples
a. Then
go to Direct Control, click
purge
manifold.
b. After
manifold is purged click under Direct Control, gel
capillary
fill
c. After
it is filled, also under the same window, hit optical
alignment
d. Then
go to Run, Monitor Baseline,
when its
dialog box appears, check enable monitor baseline
1.
If
any baselines are above 6000, try another optical alignment, or clean
off the
array, or purge the manifold
e. Then
under the Direct Control window, click Load
sample plates.
f. Load
in your sample and buffer plates in the appropriate place (Fig. 8)
g. Then
go into Run, start
sample
plate to begin your run. Make sure that the correct
cells are
highlighted
1.
The
columns each take between 90 and 110 minutes to run.
5.
Disassembly
of the CEQ8000 - after your sequences have finished
a. Remove
the sample and buffer plates from the machine
b. Rinse
the wetting tray with distilled water and refill it
c. Remove
the gel cartridge, using a Kimwipe to remove the excess gel from the
end. Replace the cartridge with the
plug.
Sequencing
checklist:
q
Prepare sample plates with sequencing
reactions.
q
Fill any wells of incomplete columns in the
sample
plate.
q
Cover all wells with mineral oil.
q
Fill corresponding wells in the buffer plate
with
buffer.
q
Create a sample plate.
Ã
¸
Export the data.
Ã
¸
Apply analysis and sequencing methods to all
wells.
q
Load the gel cartridge into the machine.
q
Purge manifold.
q
Check the baseline readings of the capillary
array.
q
Refill the wetting tray.
q
Fill out the sequencing log on the
computer.
q
Start sample plate.
Disassembly:
q
Remove the sample and buffer plates.
q
Rinse the wetting tray with distilled water
and refill
it.
q
Replace the gel cartridge with the plug.
q
Record the usage of the gel on the package and
put it
in the fridge.
BECKMAN CEQ 8000
