Pictoral Guide to Genotyping on the Ceq8000

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1 Pictoral Guide to Genotyping on the Ceq8000 The Beckman Ceq8000. available in beige

2 Index Overview Getting started Logging on to the computer Turning the Ceq8000 on The Ceq8000 software control panel The Ceq8000 database Opening the database Creating a new database Setting your database as the working database The Ceq8000 Run software The wetting tray The Event Type box Changing the gel cartridge The Ceq8000 sample sheet Running Samples in the Ceq Loading plates Putting the Ceq8000 to bed Installing the gel cartridge plug Cleaning the wetting tray Turning the Ceq8000 off

Overview: The Ceq8000 differs from the Ceq2000 because of (i) up to date software and (ii) a red and grey go-faster stripe Another satisfied customer The Ceq8000 is really very easy to use once you

3 Overview: The Ceq8000 differs from the Ceq2000 because of (i) up to date software and (ii) a red and grey go-faster stripe Another satisfied customer The Ceq8000 is really very easy to use once you have been shown! This guide is intended as a reminder only. People MUST NOT use the Ceq8000 unless they have express permission from Prof S.J. Kemp or one of his officially appointed minions. The major points to remember are: o Replace the water in the wetting tray before and after each run o Do not let the capillaries dry out a maximum of 15 min exposure to air o Use the correct sample and buffer trays o Do not run the Ceq8000 with an empty wetting, sample or buffer tray o Make sure the buffer tray lid is corectly positioned lug at front and slides freely o Replace the gel cartridge with the yellow plug when you have finished a run o Remove (or ask someone to) the capillary array if the machine is not to be used for a few days o Be gentle! IF IN DOUBT - ASK Tip: Beckman have provided many excellent guides and handouts please take time to read these as they cover the workings of the Ceq8000 in much better detail. 2

1. Getting started: Logging on to the computer

labelled 0 1 at the front of the Ceq8000.

The Ceq8000 software control panel Double click on

4 1. Getting started: Logging on to the computer Username: Administrator Password: administrator Note: the capital A on the username! Turning the Ceq8000 on Press the grey button labelled 0 1 at the front of the Ceq8000. If only the LiCor was this easy.. 2. The Ceq8000 software control panel Double click on the Ceq8000 software icon to start the software. Ceq8000.lnk Double click on this icon to get this screen 3

3. The Ceq8000 database The Ceq8000 stores all of its data in database: the size of the database is limited so that when a database is full it can be written to a CD.

Note: Selecting the correct database is your first priority since you cannot change the database when any other Ceq8000 software is running. Opening the databse click on the database icon.

5 3. The Ceq8000 database The Ceq8000 stores all of its data in database: the size of the database is limited so that when a database is full it can be written to a CD. To reduce the number of arguments, everybody is required to store their data in their own database. You will need to create your own database if you do not have one. Note: Selecting the correct database is your first priority since you cannot change the database when any other Ceq8000 software is running. Opening the databse click on the database icon. Click on this icon to get this screen If you already have created a database then skip the next step and set your database to the working database as described below. Creating a new database Use the commands: File, New Database from the menu You should see the options box below Give your database a sensible name.. Tip: use the check box to set your database as the working database when you create it. 4

Your new database will appear in the database list Note: The working databse is highlighted bold If you didn t set your newly-created database to be the working database (i.e. it is NOT bold) then do so now using the instructions below; if your database is the working database then skip the next step.

Click your database (so that it is highlighted dark blue) Right click, select Set As Working Database The working database will appear in BOLD font (see Note above).

6 Your new database will appear in the database list Note: The working databse is highlighted bold If you didn t set your newly-created database to be the working database (i.e. it is NOT bold) then do so now using the instructions below; if your database is the working database then skip the next step. Setting your database as the working database. Click your database (so that it is highlighted dark blue) Right click, select Set As Working Database The working database will appear in BOLD font (see Note above). Close database (click x in the top right hand corner or select File, Exit from the menu). 4. The Ceq8000 run software The running person icon! The Ceq8000 genetic analysis software menu appears as a toolbar after the first application (e.g. opening the database) has been run. Ceq8000 toolbar Click here! To get this message. 5

and eventually get this screen! Note: when you first start up the Ceq8000, the gel cartridge should have been removed and the Ceq8000 stored with a yellow cartridge plug in place.

7 and eventually get this screen! Note: when you first start up the Ceq8000, the gel cartridge should have been removed and the Ceq8000 stored with a yellow cartridge plug in place. The Ceq8000 refuses to recognises this and provides this warning message: Ignore this and click OK Everything you need to run the Ceq8000 can be found by clicking on the Direct Control tab. 6

8 The wetting tray The wetting tray is filled with double-deionsed water. The capillary ends sit in the wetting tray when they are not in use; this prevents the capillaries from drying out. Note: the capillaries can be exposed to air for a MAXIMUM of 15 minutes before they dry out. The wetting tray MUST be cleaned out and refilled with fresh deionised water after 12 columns (= one 96-well plate) have been run; ideally you should clean the wetting tray every time you do a new run (but not if you are testing single columns). To remove the wetting tray..click on the wetting tray icon The Ceq8000 will politely ask you if you are sure click OK to release the wetting tray The Event Type Box.. but DO NOTHING until the Event Type box says Idle. 7

The following box will appear when you are allowed to open the lid.

Note: The Ceq8000 gives you several options of where the capillary array should sit when the sysyem

Now you can open the Ceq8000 lid and remove, clean, refill and then replace the wetting tray - IN

9 The following box will appear when you are allowed to open the lid. Do NOT click anything until you have replaced the wetting tray. Note: The Ceq8000 gives you several options of where the capillary array should sit when the sysyem is idle. the default setting should be Wetting Tray. Check this DO NOT CHANGE IT. Now you can open the Ceq8000 lid and remove, clean, refill and then replace the wetting tray - IN UNDER 15 MINUTES! Open the lid The Ceq8000 lid simply lifts up...to reveal the inner gubbins of the machine. Capillary ends Position for sample tray Wetting tray Position for buffer tray and lid 8

The wetting tray is held in by two little white wings

Note: the wings cannot be pushed back if there is a

Lift the wetting tray out The lid simply lifts off

to the Ceq8000 Note: the Ceq8000 dumps acrylamide in

10 The wetting tray is held in by two little white wings which must be pushed back to release the wetting tray. Note: the wings cannot be pushed back if there is a sample tray in the Ceq8000. Lift the wetting tray out The lid simply lifts off Wash with distilled water, refill with double distilled water and replace the lid. Remember to DRY the wetting tray before you return it to the Ceq8000 Note: the Ceq8000 dumps acrylamide in the wetting tray. Always wear gloves. (The hands here are stunt hands and are dealing with distilled water only) 9

Close the Ceq8000 lid and click Done DO NOTHING until the Event type box says Idle.

11 Replace the wetting tray and push back the wings so that they hold the wetting tray in place. Tip: If you have prepared you sample tray and buffer plate you can load these while you change the wetting tray. Close the Ceq8000 lid and click Done DO NOTHING until the Event type box says Idle. Changing the gel cartridge The Life tab tells you how much acrylamide gel is left in the machine. Tip: you will need approximately 1 ml gel for every column plus an initial 1ml squirt every time you start a run (to purge the capillaries). If you need to insert an acrylamide gel cartridge, then.. Click on the gel cartridge icon 10

The Ceq8000 will very politely ask you if you are sure click OK and

Pull the door open and gently pull the cartridge holder towards you

12 The Ceq8000 will very politely ask you if you are sure click OK and the Ceq8000 will release the cartridge/plug DO NOTHING until the Event type box is Idle. Gently push the gel cartridge door inwards to release the door. Pull the door open and gently pull the cartridge holder towards you and VERY gently pull out either (i) the old gel cartridge or (ii) the yellow storage plug. Note: the cartridge/plug can be stiff and difficult to remove. DO NOT simply yank the cartridge/plug out hold the cartridge holder while you remove the cartridge/plug. 11

Remember to wipe up any acrylamide when removing an old gel cartridge!

Tip: place the gel cartridge cap onto the cartridge plug to keep it

Note: The gel cartidge and plug have wings; make sure that these fit

13 Remember to wipe up any acrylamide when removing an old gel cartridge! Remove the cap and then insert your new gel cartridge. Tip: place the gel cartridge cap onto the cartridge plug to keep it safe, you will need it to store the acrtridge. Note: The gel cartidge and plug have wings; make sure that these fit into the notches in the cartridge holder. Push the cartridge holder back into place and close the door. 12

Click the Install Cartridge button To get this screen If you are installing a NEW gel cartridge click Set to New If you are installing an old gel cartridge fill in the hours on instrument information

14 Click the Install Cartridge button To get this screen If you are installing a NEW gel cartridge click Set to New If you are installing an old gel cartridge fill in the hours on instrument information (NOT REALLY NECESSARY) When you are finished click Done The Ceq8000 will make clunking noises for a few seconds...and then tell you how much gel is in the cartridge (on the Life tab) DO NOTHING until the Event type box is Idle. Note: If you are removing a gel cartridge and there is sufficient gel remaining for further runs, please replace the cap (it should be on the yellow plug) and store it at 4 C. 13

5. The Ceq8000 sample sheet The set up icon! Click here! To get this screen! the default sample sheet. Enter your sample data in the sample columns that CORRESPOND to your sample plate setup.

It provides a reference for the sample plate. If you are running microsatellites you will need to choose Frag-3 for each column using the drop down box.

15 5. The Ceq8000 sample sheet The set up icon! Click here! To get this screen! the default sample sheet. Enter your sample data in the sample columns that CORRESPOND to your sample plate setup. Tip: You can copy and paste your sample names and plate layout from a pre-prepared Microsoft Excel spreadsheet. The Ceq8000 will put a tracking suffix at the end of the sample name leave it there! It provides a reference for the sample plate. If you are running microsatellites you will need to choose Frag-3 for each column using the drop down box. Note: When you edit the sample sheet, the edited cells turn a sickly green colour. The Ceq8000 cannot be run until the samples are blue - i.e. they have been saved. When you have finished entering your data and the columns have been given the correct run conditions (e.g. Frag-3) click on the analysis tab. 14

Until the correct size ranges, allele bins and correction for stutter/spurious bands have been set

16 Until the correct size ranges, allele bins and correction for stutter/spurious bands have been set select DefaultFragmentAnalysisParameters from the drop-down box for each sample column and tick the Automatic analysis check box.. Note: Automatic analysis is NOT necessary fragment sizing can be done at a later date using the fragment analysis software. REMEMBER to uncheck the tick box so that the default setting of AutomaticSequence Analysis is NOT set!! Save the sample sheet using the commands: File, Save as. from the drop-down menu...and your samples should be blue! You are now ready to run the Ceq

6. Running Samples in the Ceq8000 Checklist: you can run samples on the Ceq8000 if have cleaned and refilled the wetting tray,

A sample run on the Ceq8000 is begun by using the commands: Run, Start from the menu in the Ceq8000 sample set up software The run

..the following screen will appear This simply reminds you to fill the wetting tray, and which columns you should have filled on you

17 6. Running Samples in the Ceq8000 Checklist: you can run samples on the Ceq8000 if have cleaned and refilled the wetting tray, prepared your sample tray and buffer tray, and completed and saved a sample sheet. A sample run on the Ceq8000 is begun by using the commands: Run, Start from the menu in the Ceq8000 sample set up software The run software will be activated and...the following screen will appear This simply reminds you to fill the wetting tray, and which columns you should have filled on you buffer and sample trays. Note: CHECK that the highlighted columns match with your sample/buffer plates If you loaded your sample when you refilled the wetting tray then click Start Loading plates If you have not loaded your sample / buffer trays, don t panic! Simply click Load Plates.. 16

If the tray does not appear to fit, then you may be putting the tray in back-to-front Make sure that the tray fits snugly with the bottom of

18 You will get this message Click Unload To get this message DO NOTHING until the Event type box is Idle. Put the sample trays into the Ceq8000 Tip: You cannot put the sample tray into the Ceq8000 incorrectly. If the tray does not appear to fit, then you may be putting the tray in back-to-front Make sure that the tray fits snugly with the bottom of the Ceq8000. Lift off the buffer tray lid, place in the buffer tray, Tip: You cannot put the buffer tray into the Ceq8000 incorrectly. If the tray does not appear to fit, then you may be putting the tray in back-to-front 17

You can go to the Log to check that the Ceq8000 is running, Note: If there is just enough acrylamide gel to run you sample the Ceq8000 will check the amount of

19 put the buffer tray lid back it should SLIDE freely CHECK THIS! Note: the front lug points towards you! and then close the lid. Slide freely Once the sample and buffer trays have been Loaded, click load DO NOTHING until the Event type box is Idle. Now click Start to begin the run. You can go to the Log to check that the Ceq8000 is running, Note: If there is just enough acrylamide gel to run you sample the Ceq8000 will check the amount of gel detected.. Note: If there is not enough gel to run the full sample plate the Ceq8000 will give you the option to o run the columns until the acrylamide has been used up or o abandon the sample plate. 18

7. Putting the Ceq8000 to bed. More DETAILED instructions can be found in the Changing the gel cartridge and The wetting tray sections of this guide.

20 7. Putting the Ceq8000 to bed. More DETAILED instructions can be found in the Changing the gel cartridge and The wetting tray sections of this guide. The software process is mostly the same (differences are shown below) but the manual bit is backwards. Installing the gel cartridge plug When the Ceq8000 has completed its run, simply click on the gel cartridge icon on the Direct Control tab to release the gel cartridge Clean up any acrylamide when removing the gel cartridge and INSERT the yellow plug remembering to match the wings of the plug with the notches of the gel cartridge housing. Click on Install Plug when you have finished DO NOTHING until the Event type box is Idle. Cleaning the wetting tray Next click on the Wetting Tray icon to release the wetting tray. Note: you will have to remove your sample tray to clean the wetting tray Tip: when you clean the wetting tray remove the buffer plate at the same time! Do not forget to make sure that the buffer tray lid can move freely and to position the lug forwards. Exit the Ceq8000 software by selecting File, Exit from the menu. Turning the Ceq8000 off Click Exit from the toolbar. As usual, the Ceq8000 will ask if you are sure. Click Yes Turn the Ceq8000 off by pressing the grey button at the front. 19

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