ABI 377 & ABI 3700 DNA Sequencers

Introduction
This SOP describes how to set up plates, and prepare a gel for a sequencing 377 run.

Materials

10% Ammonium Persulfate (see SOP/HH5TA02/1998/0046)
TEMED
Accugel
Glass beaker
25 ml stripette and electronic pipetter.
377 glas plate pair [Ask Jackie, these are specially etched]
377 0.2 mm spacers
377 0.2 mm sharks tooth well former comb
Black Cassette

Method
For 1 Gel - Accugel

30 ml acrylamide
150 µl 10% APS
 21 µl TEMED

Preparing the plates

* The glass plates for the 377 machines are long plates with small indents at the base of the back plate, and notches at the top of the front
plate
* The plates are produced in pairs and must stay within these pairs, each plate also has an inside and an outside.
* The outside can be identified as all the plates are marked with either a manufacturers stamp or with an identification number written with a diamond pen, some may even had OUT written on the outside.
* The spacers for the 377 gels are long with a notch at the top.  This notch faces the inside of the gel.

1) Place the bottom plate in cassette, making sure that the notched ends are against the silver locating pins.  Wipe the spacers before use, then place them down the long side of the back plate, then put the top plate on and clip all but the top clips in place.

2) Slide the black clamp onto the cassette and under the glass plates.  Tighten the white plastic screws so that the black clamp bends a little at each end. [it is important to tighten these screws as otherwise the comb will not be tight in the gel and samples will leak.

3) Make up the gel mix as above, in clean glassware, using the electronic pipette to mix it by pipetting slowly up and down, taking care not to introduce bubbles.  Add the gel mix slowly with an electronic pipette to the top of the gel, tapping on the top of the glass plate at the gel front to stop bubble formation.

4) Once the gel has filled the plate insert the comb. These are found in the racks next to the sink. The flat edge of the sharks tooth is placed into the gel with the teeth sticking out the top.

5) Clamp the gel by the comb with the transparent clamp and clip the two clips on the cassette.

6) Allow to set for 2 hours before use.  If the gel is to be left for a long period before use it must be wrapped, top and bottom, with Kimwipes soaked in 1xTBE, then cling film.

7) The gels are numbered sequentially to allow tracking in the event of any problems and logged into the gel book in the gel pouring room.

Safety Issues

Unpolymerised acrylamide is a powerful neurotoxin and putative carcinogen.  It should always be handled with gloves and eye protection must be worn to prevent ingress by splashing to eye.  Refer to COSHH form WTCHG97-24. Polymerised acrylamide cannot traverse skin but gel matrix always contains small amounts of monomer so gloves should be worn whenever handling gels.

USE OF AN ABI 377 FOR SEQUENCING

Introduction

This SOP describes how to use an ABI 377 for sequencing.

Materials

377 sequencing gel (SOP/QH58Z02/1999/0062)
1xTBE (SOP/QH58Z02/1999/0027)
Sequencing Samples

Method

1) Preparing the machine

After the previous run is completed remove the heating plate and place handle side down on the bench [DO NOT PLACE FLAT SIDE DOWN AS THIS CAN RUIN THE HEATING PLATE]. Remove the used cassette and take it complete with the upper buffer chamber to the sink.Take off upper buffer chamber and wash with RO water before use. Take used gel out of the cassette and place in tray provided ensuring that there is tissue between the top of the gel already in the tray and the gel to be put in.  This prevents scratching of the glass plates.  Wash the cassette with RO water and dry before use.
Discard buffer from the lower buffer chamber and wash with RO water.
Collect a  sequencing gel  from the gel pouring room , wash off  excess acrylamide with warm water followed by deionised water and dry plates using white kimwipe, lint free tissues (red box).
Take out the pouring comb and make sure there is no acrylamide left in-between the gel plates at the top.
Clean between the plates with an old loading comb to remove any  of the old acrylamide.
Wash the well left by the sequencing comb with 1 x TBE  and carefully insert the sharks tooth comb into the gel so that the tips of the teeth just penetrate the top of the gel.
Do not push the comb into the gel too far otherwise it will be difficult to load the gel.
If the comb has been pushed into the gel too far but is still loadable DO NOT remove the comb and try again as this will ruin the wells.
Clip the gel into the ABI cassette ensuring the gel plates are flat in the cassette.

2) Preparing the computer

Before each run, restart the Mac computer by going to the Special menu and selecting Restart.  This ensures that the RAM memory is not fragmented and will restart the collection software.
Select ABI Collection Software, this nornmally comes up automatically when the computer is restarted.  In the File menu select New then select Sequencing Run.

In the Run window that appears make sure that the following settings are present.

For dRhodamine and BigDye Terminators.

PLATE CHECK: Plate Check A.
PRE RUN: Seq PR 36E-2400
RUN: SEQ RUN 36E-2400.
MATRIX:  ABIX_BDT_ACCUGEL_DEC99

For the matrix setting the X refers to the machine so ABI A will have a matrix called ABIA_BDT_ACCUGEL_DEC99

[Note: On the Monaco ABI the pre run and run modules have the suffix chiller added because of the additional water bath installed]

To change the number of lanes run select either 36 and full scan or 48 and XL scan.

Plate Check
Press plate check and then the scan window will appear and after approximately 1 min 4 coloured lines will appear. These should be flat and  below 2048.  If there are peaks in this trace this indicates that the plates are not clean and the cassette needs to be taken out of the machine and cleaned with kimwipes again. If the peak is not removed the corresponding lane may have to be missed out during loading.

If the plate check is OK

Clip on the buffer chamber and fill with 1 x TBE. Wait a minute to ensure there is no leakage of buffer down the front of the plate. This could cause arching of electrical current if left and severely damage the ABI.
Next clip on the heating plate and fill the lower buffer chamber with 1 x TBE.
Rinse out the wells thoroughly to ensure that there are no air bubbles or excess acrylamide present.

Pre Run
Select pre-run. Then go to Window: Status and check the run is for 1 hour and that the gel gets up to temperature 51^oC.
You should allow at least 10 minutes for the pre-run.
While the gel is pre running prepare the sample sheet by selecting File - New -Sequencing Sample sheet and typing in the appropriate lanes. Save the sample sheet.
Prior to loading your samples and performing a run, add 3 µl loading dye for ABI377. Make sure the pellet, which is not visible, is thoroughly resuspended either by pipetting the loading dye up and down the tube a few times or by vortexing gently for 10-15 seconds to ensure that your sample is resuspended in the loading dye. Centrifuge samples briefly.
Denature the samples for 2 mins @ 95 ^oC and immediately store on ice until ready to load (this is important as DNA will re-anneal if not immediately placed on ice).
Pause the Pre Run and wash out the wells once more.
Load sample sheet into the run window [ the machine will not be able to start a run without a sample sheet]
Load 1-2 µl per lane, loading every other lane and then cancel the pause- press continue - and run the samples in for 3 minutes.
Pause the gel again and wash out the wells and load the well not loaded in the first round.  Run the samples in for a further 3 minutes and then cancel the Pre Run.  Start the Run and then a window will appear asking to name the run.
After the run has finished the run folder can be transferred to another computer for further analysis

[The reason for loading every other lane is that the machine can recognize the individual  lanes and track the gel in the analysis program.  If every lane is loaded at the same time with a sharks tooth comb the machine will only see one broad lane]

PREPARATION OF A SEQUENCING SAMPLE SHEET

Introduction
This SOP describes how to produce a sample sheet for sequencing.
 

Materials

List of samples.

Method
373  e.g. ABI 11  In the File menu select New Sample sheet.  Fill in the Sample file name then the Dye Set/Primer name.  For BigDyes this is "373 BDT" and for dRhodamine this is "373 dRDT".  There are 4 choices which can be selected under the heading Auto Settings ? AA = auto analyse, CS = contains sample, PR = plot raw data, PA = plot analysed data.

373-XL and 377  e.g. ABI 10  In the Data Collection Software select File then New then Sequence Sample.  Fill in the Sample name then the Dye Set/Primer name.  For BigDyes this is "373 BDT" and for dRhodamine this is "373 dRDT".  Select the appropriate matrix, this must be done if automatic analysis has been selected.

Safety Issues
None.
 
PRODUCTION OF SEQUENCING MATRIX USING DATA UTILITY

Introduction
This S.O.P. describes the production of a sequencing matrix on both 373 and 377 ABI machines.

Materials

373 or 377 gel (see SOP/HH5TA02/1998/0023 and SOP/HH5TA02/1998/0010)
BigDye dRhodamine matrix dye set
Deionised Formamide

Method

Set up the gel in ABI machine as SOP/QH58Z02/1999/0066, SOP/QH58Z02/1999/0067 or SOP/HH5TA02/1998/0012, for ABI373, ABI 373-XL and ABI 377 respectively.

Combine 2.5 µl of each matrix standard with 2.5 µl deionised formamide, in duplicate.  This will give you 8 samples, 2 of each dye type.  Heat each sample to 96^oC for 2 minutes to denature before loading.

Create a sample sheet to assign sample (see SOP/QH58Z02/1999/0064) but do not assign a matrix to the samples and do not allow autoanalysis.

Load the Matrix Standards in duplicate, 2.5 µl of the matrix standards, i.e. load 8 lanes 2 dR110, 2 dR6G, 2 dROX and 2 dTAMRA, leaving an empty lane between each standard.  Select the appropriate filter-set, and start the run.  377 ? Run modules containing filter set E, 373 ? filter-set A.

After the gel has run
Open up Sequence Analysis software
Go to File, then Open, then Collection gel and open the gel you wish to produce a matrix from.

Track the lanes containing the Matrix standards - this is done by selecting a lane and going to Gel on the menu and selecting Mark lane used then move the tracking lanes so that they follow exactly the lane of that well.  When this has been done for all the lanes used, go to Gel and Extract the lanes.

From within the Utilities folder of the Sequence analysis software, open the DataUtility program.  In the Utilities menu, choose Make Matrix.
In the dialogue box that appears make sure that the Dye Primer matrix button is selected.  In the next box that appears press the "C" nucleotide button and select the appropriate sample file for that nucleotide according to the table below.

Box Dye Primer Matrix Taq Terminator Matrix T7 Terminator matrix
 
 

For each sample fill in a start point, where analysis of each sample will start, start with the default value of 2000 and for the points choose 1500, for the number of data points to analyse.

Click New File and name the new matrix e.g. BigDye ABI10 Oct99.
Then press OK.

The computer will then try and make the matrix, if it is unable to do this an error box will appear telling at which point it has failed.  One solution if it has failed to make a matrix is to increase the amount of data points to analyse.

If the matrix has been successful go back to the Utilities menu, choose Make Matrix.  This time make sure that the Taq Terminator button has been selected before choosing the sample files.  After selecting the sample files, select Update the File and choose the correct matrix name e.g. BigDye ABI10 Oct99 to update.

Repeat again as above but this time making sure that the T7 Terminator Matrix button has been selected.

The matrix is automatically saved in the ABI folder.

To check the matrix, go to the utilities menu and select copy matrix.  Under Source, select Instrument file and choose the matrix you wish to check.  The 3 matrix files are all shown at once and should look like the box below.