Table of Contents
Chapter 1. User Information and Terms of Use
Chapter 2. Center Use Guidelines
2.1: Personal Safety
2.2: Cleanup
2.3: Reagents
2.4: Accidents
2.5: Equipment Use
Chapter 3. Expected Level of Experience
Chapter 4. Primer Information
4.1: Selecting/Screening Primers
4.2: Dye Set/Filter Set to Use
4.3: Multiplexing
5.2: Server Access
5.3: Running a Gel [1]
Chapter 6. Analyzing Your Gel File
6.1: Using GeneScan to Work With Your Gel File
6.2: Using Genotyper to Call Alleles
Chapter 7. Hydra 96 Liquid Handler
7.1: Overview
7.2: Operation
7.3: Startup Procedure
7.4: Shutdown Procedure
Chapter 8. Recipes
8.1: 10x TBE
8.2: 1x Gel Buffer
8.2: Polyacrylamide Gel Solution
8.3: Loading Mix
Table of Contents
Chapter 1. User Information and Terms of Use
Chapter 2. Center Use Guidelines
2.1: Personal Safety
2.2: Cleanup
2.3: Reagents
2.4: Accidents
2.5: Equipment Use
Chapter 3. Expected Level of Experience
Chapter 4. Primer Information
4.1: Selecting/Screening Primers
4.2: Dye Set/Filter Set to Use
4.3: Multiplexing
Name: ______________________________________
Campus Address: _____________________________
Phone Number: _______________________________
E-mail:______________________________________
Account_____________________________________
The Illinois Genetic Marker Center hereafter referred to as “center” presents the following terms of use to the above named person hereafter referred to as “user.”
1. The user will schedule the use of each piece of equipment with the center using the online scheduling system.
2. The user will have completed training for each instrument before using it. This includes but is not limited to thermocyclers and DNA sequencing machines, and could extend to centrifuges, pipetmen, gel rigs, balances and computers if the director of the center deems such training necessary.
3. The user will exercise care in the use and maintenance of center equipment. The cost of repair or replacement of equipment due to damage caused by the user's actions or neglect will be paid by the user.
4. The user has received and will follow the Center Use Guidelines.
5. The user will keep a record of the procedures performed (i.e. ran one 377 gel) and supplies used (i.e. 1 sample comb, 4 boxes of plates) as directed by the center.
6. The user will be billed for center use at the rate indicated in the center fee schedule at the time of use. (May change without notice as services change or are added.)
7. A setup fee may be required to cover initial costs.
8. Computer use:
a. Each user will be assigned a computer account and password.
b. User will not share their password or accounts with others.
c. Each user will be allowed to use up to 5 Gb of network storage space. The system administrator will back up said data space according to a regular backup schedule. (1.7.30 archive) Users are responsible for keeping below the 5 Gb limit and may move data to a CD-R.
9.
The Center will be open for use between the hours of
I ______________________________ the undersigned
have read and agree to abide by the above terms of use for the
_______________________________________________ Signed Date:
Table of Contents
2.1: Personal Safety
2.2: Cleanup
2.3: Reagents
2.4: Accidents
2.5: Equipment Use
1. No food or drink of any kind will be brought or consumed in the laboratory.
2. Wear appropriate clothes.
3. Gloves and protective eyewear must be worn whenever laboratory procedures are being carried out.
4. Tie back loose hair and clothing, and remove any loose jewelry during laboratory work.
5. Horseplay in the laboratory is strictly prohibited.
6. Broken glass should be put into the sharps container, not the regular trash.
7. Know the location of the fire extinguisher, eye wash, and safety shower in the lab and know how to use them.
8. Know the building evacuation procedures.
1. Clean up after yourself. All users are responsible for maintaining cleanliness in the laboratory and properly disposing of consumed chemicals or other experimental refuse.
2. Maintain a clean and orderly laboratory bench and drawer. Keep aisles clear by keeping backpacks, coats, etc. in provided space. Clean up and put away all glassware and equipment before leaving at the end of a laboratory session.
3. Wipe up all small spills on reagent tables, balances, counter tops and hood areas immediately. Small spills containing less than fifteen grams of solid or less than fifteen milliliters of liquid. Such spills can usually be cleaned up with paper towels. Take care to avoid skin contact with chemicals during clean up.
4. Keep working area tidy and clean.
5. Dirty containers, tools or equipment must be cleaned immediately.
6. Wipe off each piece of equipment and bench top when you are finished with it (not scrub, just wipe).
7. All benches and sinks must be thoroughly cleaned at the end of each laboratory session.
8. Stools will be placed under the work bench at the end of each laboratory work period.
1. Avoid direct contact with all reagents. Do not inhale vapors, ingest reagents, etc.
2. Never hold bottles by the neck.
3. Make sure that the name and concentration on the label of the bottle are exactly that which you require.
4. Reagents specific to your research should be stored in clearly labeled bottles identified by your name, date, contents, and any relevant cautions.
a. Labels should be firmly attached.
b. Any non-labeled items will be destroyed.
c. Bottles should be stored in the appropriate reagent storage location.
5. Prevent contamination of stock reagents, because these materials are shared by all users of the facility.
a. Never return any reagent to a stock bottle.
b. Do not introduce a sampling device, such as a spatula or pipette into a reagent bottle unless directed to do so.
6. When pouring a solution or liquid, do not place the stopper on the bench top. Instead, hold the stopper between the fingers while dispensing the liquid and then replace the stopper immediately when finished.
7. Dispose of solid materials such as filter paper, indicator paper, weigh boats, and insoluble solids in the appropriate waste containers or trash can. Do not dispose of such items in the sinks. Dilute water soluble liquid wastes with running water while pouring them into the sink drain unless instructed to place them in a designated waste container.
1. All laboratory accidents (spills, cuts, burns, etc.) must be reported to the director immediately, regardless of how trivial they seem.
2. If you spill a reagent on your skin, wash the reagent off immediately with plenty of cold water (use the emergency shower or eye wash station as necessary).
1. Know proper use of equipment before using.
2. All critical equipments have log books. Each user will sign in the log giving the date and time he or she uses the equipment and other requested information.
3. In the event that equipment fails to perform correctly, immediately notify the lab staff and note problem in log book. Do not attempt repair yourself. Leave a note on the white board.
4. Materials and instruments must be in workable condition.
a. Wet electrical devices must not be used.
b. Defective instruments have to be withdrawn from use and their condition indicated.
5. All broken or damaged equipment must be reported to lab staff. If you are in doubt as to the condition of the equipment, notify lab staff immediately. Missing equipment should also be reported to the lab staff.
6. Breakage of equipment due to negligence will be charged to the user at replacement cost.
7. Equipment will be cleaned and returned to its designated spot when not being used.
In order for the center to operate efficiently for everyone, we must expect a certain level of competence in our users. Before starting work in the center, users must have mastered basic laboratory skills such as pipetting, calculating concentrations, mixing solutions, PCR and electrophoresis. Additionally, they must be sufficiently versed in theory such that they can troubleshoot their work. The director of the center reserves the right to screen users to assess a potential user's abilities as well as to revoke the access privileges of any user who does not perform to the expected level of competence. While we do not expect users to know how to operate the equipment in the center without training and understand that unexpected situations will occur, we must expect the users to be able to work independently after training so that we can continue to operate the center with a small staff in the most economical way.
Table of Contents
4.1: Selecting/Screening Primers
4.2: Dye Set/Filter Set to Use
4.3: Multiplexing
Due to the fact that the Illinois Genetic Marker Center does not restrict use of the facility to any specific species, we cannot be experts on primer selection. It is up to the individual researcher to choose primer pairs which are most informative for the project on which they are working. As part of the training process, users may elect to screen their primers in the Center. This can be done in conjunction with determining the appropriate dilution rate for the user's PCR products and the construction of a specific matrix file. Once the primer pairs have been chosen, it is useful for the researcher to sort them based on relative fragment size. This information will be used to determine which dye to attach to a given primer and how to multiplex the PCR products.
The ABI Prism 377 DNA sequencing machines that we use in our lab are fitted with virtual filter sets A, C, D and F. Different filters allow you to use four different dyes. With Filter A you can use dyes TAMRA, HEX, 6FAM and ROX with ROX as the size standard. With filter C, you can use dyes 6FAM, HEX, TET, and TAMRA with TAMRA used as the size standard. Dyes ROX, 6FAM, HEX, and NED can be used with filter D with ROX being the size standard and this is the most common filter set used at the center. Finally, with filter F you can use dyes 5FAM, JOE, NED, and ROX with ROX being the size standard.
In order to get the most data for a given cost, the researcher can combine PCR products with different dye tags and different fragment sizes in a given lane. In the most basic sense, the 377 can sort by a combination of size and color. When considering which dye should be attached to which primer, one should choose different dyes for those with similar fragment sizes. The same dye can be used with primer that produce fragments of significantly different sizes. At minimum a researcher can multiplex by three (by having 3 dyes) in each lane. Careful planning will allow multiplexing to an even greater degree. Because we must charge based on the actual supplies used, and there is a fixed cost per gel, the more a researcher can multiplex, the lower the cost of data production.
Table of Contents
Chapter 5. Genotyping
5.2: Server Access
5.3: Running a Gel [1]
Chapter 6. Analyzing Your Gel Data
6.1: Using GeneScan to Work With Your Gel File
6.2: Using Genotyper to Call Alleles
Chapter 7. Hydra 96 Liquid Handler
7.1: Overview
7.2: Operation
7.3: Startup Procedure
7.4: Shutdown Procedure
Chapter 8. Recipes
8.1: 10x TBE
8.2: 1x Gel Buffer
8.2: Polyacrylamide Gel Solution
8.3: Loading Mix
Table of Contents
5.2: Server Access
5.3: Running a Gel [1]
5.3.1: Plate assembly
5.3.2: Pouring a gel
5.3.3: Making a sample sheet
5.3.4: Preparing your samples
5.3.5: Performing a plate check
5.3.6: Loading the samples onto the gel
5.3.7: Transferring the gel file to your server
5.3.8: Taking down the finished gel
5.3.9: Plate separation and cleaning
The workflow in the center can be broken into three main parts.
1. PCR amplification using selected primers.
2. Electropheretic separation of fragments using the ABI Prism 377 DNA sequencing machine.
3. The analysis of the data produced by the ABI Prism 377.
This part of the User Guide will cover the day to day procedures and equipment used in the lab.
The very first thing that needs to be done is to sign up for the ABI 377 machine on the website (http://genotyping.uiuc.edu). This needs to be done to be sure that you have a machine reserved for the time you need to run a gel and clean it up. If you don't have it reserved, you can't use it.
To log onto your server account, use the following procedure.
· Click on the Apple icon on the desktop. Click on "Chooser"
· In the Chooser window, click on the "Apple Share" icon. Click on "Server IP Address…" key on right.
· In the "Enter the Server Address" prompt window, type in "genotyping". Click on "Connect".
· In the "Connect to the file server "genotyping" as" prompt window, type in your userid and your password. Click on "Connect".
· A prompt widow will appear asking you if you want to open your server account; hit "OK".
· Your server folder should now appear on the desktop.
· When you are ready to log out of your server account, drag your "Server Folder" icon into the "Trash".
To assemble the plates to run a gel, use the following procedure.
· Clean plate thoroughly and wipe with Kimwipes to get rid of lint.
· Using an inverted Styrofoam tube tray, place the back plate (no rabbit ears, side notches at bottom) with inside surface up.
· Place 3-4 small drops of water along the edges of the gel spacers and place them on top of the plate (water will hold spacers in place). Spacers should be flush with the edges of the plate, and should cover the removed notches at the bottom. Lightning bolt cut-outs should be towards the inside of the plate.
· Place upper plate on top, making sure that the gasket mark is facing out (you should be able to feel the hydrophobic region with your finger).
· Place 3 medium sized binder clips along both long edges with clamp in the center of the spacers.
The next step is to pour the gel you will need.
1. Make the initial gel solution using the following recipe.
Polyacrylamide Gel Solution (4.8%)
o 14.4 g Urea (at least electrophoresis grade)
o 4.8 ml 40%; (19:1) Acrylamide/Bisacrylamide
o 22 ml ddH2O
o 1 g Amberlite Resin
Mix all ingredients in a beaker and stir until solution is clear (10 minutes minimum). During this time the resin will grab ions present in the solution. Filter solution through a 0.2 micron filter, and degas for 5 minutes by leaving vacuum on. Bring to 36 ml with ddH2O, store in dark until ready to pour gel. Solution is stable for 48 hours.
2. After the plate is assembled and the gel solution is finished stirring, you are ready to pour the gel.
3. Filter out the deionizing beads using a syringe filter and a 60 cc syringe.
4. Add 4 ml of filtered 10x TBE to the gel solution.
5. Add 200 μl of thawed APS (this is already in small aliquots).
6. Add 25 μl TEMED (steps 5, 6, and 7 must be done in the stated order.)
7. Swirl gently to mix the solution thoroughly. After you add these three reagents you can't wait to pour the gel because it will polymerize quickly.
8. Quickly draw solution into a 30 or 60 cc syringe and cast between plates by moving syringe back and forth between plate "ears" continuously, evenly dispensing acrylamide. At the same time, tap the plates just in front of the moving gel (especially along the slower moving sides) to ensure that bubbles do not form and to help move the acrylamide to the bottom of the plates.
9. After gel mixture completely fills plates, insert casting comb into top of glass plates (do not clamp the comb down).
10. Allow gel to polymerize. This can take as little as 20 minutes, but an hour polymerization time is recommended. (A gel can be poured as much as 24 hours before it is to be used.)
11. While the gel is polymerizing, you can turn the ABI 377 and computer on to warm up and prepare your samples for loading.
The sample sheets made using this procedure are applied to the gel when beginning the run.
· Open "ABI Prism™ 377-96 Collection" software.
· Select "New" under the File menu (QK = OA N).
· Click on "GeneScan™ Sample".
· Under the "Sample Name" column, type the name of the sample to go in each lane.
· Leave "Project Name" blank.
· In the "Std" column, click on the cell next to the Red color and use the Edit menu to "Fill Down" for each sample to signify that the red is the size standard.
· Under "Pres" column, click on the appropriate box(es) next to color(s) to designate which color(s) is present, and use the Edit menu to "Fill Down".
· Type the name of each primer next to corresponding color in the "Sample Info" column. Separate primer name with slashes if more than one primer per color. Use the Edit menu to "Fill Down" the rest of the rows.
· In the "Comments" column, it may be helpful to type in some information about the sample set.
· Save the sample sheet by selecting "Save As" from the File menu. Often it is helpful to name the Sample Sheet with a date.
· Close the sample sheet file.
If the sample sheet is not on the computer that you will be running the gel on, transfer the file to the server and then off of the server onto the computer you are using.
Take the appropriate plate out of the freezer. The samples will need to be diluted (frequently 1:4 will work, but the dilution depends on PCR yield).
1. Take a reusable plate and place it in a plate holder.
2. Place 3 μl of water in each well of the plate.
3. Get the appropriate loading size standard mix out of the refrigerator to prepare the loading mix using the following recipe.
Loading Mix (100 samples)
o 250 μl deionized formamide
o 25 μl GeneScan-500 size standard (ROX)
o 25 μl Loading Buffer
Vortex to mix and store at either -20ºC or 4ºC.
4. Put another reusable plate in a holder and put 3 μl of the loading mix in each well using the repeater.
5. You will now use the Hydra to add the samples to the appropriate plates. Section 8 tells how to use the Hydra.
6. To add the samples from the PCR plate to the dilution plate, use File 9 on the Hydra.
7. To add the diluted samples to the loading plate, use File 8 on the Hydra.
8. Be sure the spin the plate down after each addition with the Hydra to ensure the entire sample is at the bottom of the wells in the plate.
9. After the diluted samples are added, put a cover on the plate and place it in the Tetrad. Select the "Denature" program on the Tetrad to denature the product.
10. Cleaning the gel plates for the run
11. The gel should be polymerized by now so it can now be cleaned. Remove all the clamps and well forming comb. Clean the comb region with a squirt bottle of ddH2O to remove small pieces of acrylamide that accumulate in the region. The little piece of spacer can be used to help clean the residue out of the well. Clean off any residual acrylamide from the external surface of the plates. Be sure the lower five inches of the plate are especially clean so that the laser does not pick up stray fluorescence.
12. Load the glass plates into the cassette by placing the cassette on a clean surface and sliding the plates in until the back notches catch on the metal plate stops. Turn clamps along cassette to lock glass plates into position. Verify that the read region (under the laser safety bar) is clean.
13. Install the lower buffer chamber and then mount the cassette with glass plates into the 377, turning the large clamps to lock cassette into place. Add 1x TBE to lower buffer chamber.
14. Now would be a good time to put the samples in the Tetrad to denature them. This way they should be done about the same time you are done with the plate check.
Certain steps have quick key strokes that are easier and will help save time. After a step that can be performed using a key stroke, you will see QK = OA followed by a letter. QK signifies there is a Quick Key stroke and OA signifies the Open Apple key which should be held down while the next letter key is pushed.
· Next open the Collection Software on the computer.
· Prepare for run by clicking "New" under the File menu (QK = OA N).
· Click on "GeneScan Run"
· Select the appropriate Plate Check file. (The letter of the plate check corresponds to the filter you are using. To see what filter to use, refer to section 13)
· Next, select the appropriate Run Module. You need to select it for the length of the plate, filter, and the voltage the gel will be run at. EXAMPLE: "GS Run 36D-2400"; GS (Gene Scan) 36 (36 cm plate) D (filter D) 2400 (2400 scans per hour).
· Next, be sure the time is set for 2.5 hours and the plate length is set for 36 cm. Also, be sure the number of lanes is set at 96.
· Select the Sample Sheet that you created previously (see section 10) by clicking on the arrow next to the sample sheet bar.
· Find the appropriate sample sheet and click on "Open"
· Under the Matrix File, select <none> on the first sample.
· Fill all rows by highlighting the entire matrix file column and selecting "Fill Down" under the Edit menu (QK = OA D).
· Also, turn off the Auto Analyze by clicking on the "X" in the Auto Analyze column and fill all lanes using the same fill down method described above.
· Hit the "Plate Check" button at the top to perform the plate check.
· When the colored lines appear on the fluorescence display window, be sure every color shows a straight line (no peaks). The green fluorescence should be the lowest line and the red should be the highest. This all shows the plate is clean. Small peaks may run themselves off the gel because they may be defects in the bottom of the gel. If the peaks don't disappear, take the gel plates out, clean them again, and check the plate again. If the peaks don't go away, a new gel needs to be poured.
· Once the plate check is finished, "Cancel" button and then "Terminate" the run.
· Now install the top buffer chamber and fill it to the fill line with 1x TBE.
· Attach the hot plate. Be sure to check the O rings on the clamps before attaching the tubes.
· Your samples should now be done denaturing so you can remove them from the Tetrad and load them on the gel.
After removing the denatured samples from the Tetrad, you will need to load them onto the gel using the following procedure.
· Using the multi-channel pipette, load 3 μl of each sample into the loading tray. The multi-channel can be used to load the first 80 samples in every fifth well and then the last 16 have to be loaded individually.
· Dip the membrane comb into sample loading tray for 10 seconds. Do not leave the comb in the tray for more than 10 seconds, doing so will weaken the comb and make it difficult to insert into well.
· Remove Comb from tray and immediately insert comb on top of gel. The teeth of the comb should barely contact the top of the gel surface or rest a few millimeters above the gel surface.
· Select "Run" on the computer to start the run.
· A box will appear asking you to name the file for the run. Be sure to choose a name that explains what is in the gel including the samples and the markers used.
· After naming the file, hit the "Save" button. The gel will now start to run.
· Go to the Windows menu and select "Status" to open the status window for the gel that is running. The amount of time remaining for the gel run is displayed in this window.
· The gel will now run for 2.5 hours.
· Be sure all of the materials you used to pour the gel, prepare the sample, and load the gel are cleaned up and washed. Do this now rather than waiting until the gel is finished running so that the communal work area is ready for the next person to use.
When the gel is finished running, transfer the gel file to your server folder using the following procedure.
· When the gel is finished, log onto your server account (see section 5.2).
· Select the gel file on the computer's hard drive and transfer it to your server folder.
· Once the gel file has been moved, you can shutdown the computer.
· Turn off the ABI 377.
After you have transferred your gel file onto the server, take the gel down using the following procedure.
· Remove the lid from the upper buffer chamber. Using a small beaker, remove three-quarters of the buffer in the upper chamber so that the buffer level is below the gasket seal.
· Remove the hot plate (if you are using a hot plate).
· Remove the top buffer chamber being careful not to spill the remaining buffer.
· Remove the cassette from the machine.
· Remover the bottom buffer chamber, again being careful not to spill any of the buffer.
· Now wash all of the parts using hot soapy water followed by a deionized water rinse.
· Lay the gel plate flat on the bench.
· Take the plastic wedge in one hand and place the other hand on top of the plates. Insert the edge of the plastic wedge between the two plates along the side pressing it in until it separates. Do not twist the wedge as this is not very effective and will ruin the wedge. Never try to pry the plates apart at the ears at the top of the plate. This can cause them to break off and then the plates can't be used again.
· After the plates are apart, place a paper towel over each of them to pick up the gel off of the plates.
· Wash plates with very hot water and Alconox solution. Do not use ethanol or any other detergent. If you do residual fluorescence may impede sample detection. Also be sure to use a soft sponge so you don't scratch the plates.
· Rinse with very hot water, then with deionized water.
· Wipe down with paper towel and allow to air dry completely.
Table of Contents
6.1: Using GeneScan to Work With Your Gel File
6.1.1: Transferring gel file to computer
6.1.2: Installing gel matrix
6.1.2: Tracking lanes (Automatic Tracking)
6.1.3: Manually editing tracked lanes
6.1.4: Extracting lanes
6.1.5: Analyzing lanes
6.2: Using Genotyper to Call Alleles
6.2.1: Opening results file in Genotyper
6.2.2: Making a Genotyper category
6.2.3: Calling alleles
6.2.4: Creating and exporting tables
6.2.5: Checking the exported tables
In order to track lanes, you will need to transfer the gel file to the hard drive of the computer using the following method.
· Log onto the server from your computer (see section 5.2).
· Open your Server Folder.
· Find the gel file you want to track.
· Open the hard drive of your computer and find the file you want to put the gel file in.
· Transfer the gel file from your Home Directory to your computer's hard drive
· Open the gel file that you want to track.
· Choose “Install New Gel Matrix” from the Gel menu.
· Choose the matrix file based on the sequencer and type of gel used.
· Click “Regenerate Image” in the prompt window.
The lanes of the gel need to be tracked so the correct sample label can be assigned to the correct lane and so the alleles can be assigned to the proper samples. The following method can be used to track the lanes.
· Choose “Track Lanes…” from the Gel menu. Click on “Auto-Track Lanes” when the prompt window appears.
· Look at the last line of the Analysis Log window. Look at the lane assignment confidence value given at the bottom of the Analysis Log window. If it is low, you will need to carefully edit your tracked lanes. If it is high, hopefully you will have little tracking to do.
· You can zoom in and out to see the individual lanes better. To zoom in, select "Zoom In" under the View menu and to zoom out, select "Zoom Out" under the View menu (QK = OA + and OA -, respectively). You will want to zoom in as close as possible to see each individual lane clearly to ease the tracking.
· The on screen color intensity can be adjusted to see the bands more easily. (Note: this does not affect the peak height during data analysis.) To do this select "Adjust Gel Contrast" under the Gel menu (QK = OA J) and move the top triangles for the appropriate color. You will want to turn the intensity of the red very high (move the top red triangle to the bottom) so it will be easy to track.
· Track each lane manually by moving the diamond notches on the line near the bands. Be sure that the line goes directly through the center of all the bands, especially the red.
· Be sure all the diamonds at the top of the gel picture are white.
· If all aren't white, highlight the ones that aren't. Click on "Mark All Lanes For Extraction" from the Gel menu.
· Extract all the lanes by selecting "Extract Lanes…" from the Gel menu (QK = OA L)
· When the box appears, uncheck the box next to "Auto Analyze New Sample Files"
· Hit the "OK" key.
After all the lanes are extracted, an "Analysis Control" window will appear.
· Click and hold the arrow next to the "Size Standard" column. Choose Define new. Assign the appropriate size to each peak.
· To save your "Size Standard" file, click on File and choose Save As and name the file.
· Click on the “Size Standard” column header in Analysis Control window to highlight the entire column. Click on the right arrow and choose the file you just made. This is a “fill down” shortcut.
· Click and hold the arrow next to the "Parameters" column. Define new. Choose defaults. Click on "Save As" under the File menu and use this to analyze the gel. Use the fill down shortcut described above to fill all rows.
· In the Analysis Control window, highlight any or all the colors to the left of the sample names that you want to analyze by clicking on each of the color squares at the top of each column.
· Hit the "Analyze" key at the top.
· Review "Analysis Log" from the Windows menu.
· Review "Results Control" from the Windows menu.
· When the analysis is done, be sure to save this new GeneScan file by selecting "Save Project As" under the File menu. Put it in your server folder and hit "Save as".
In order to call alleles in Genotyper, you will need to move the GeneScan file on to your computer using the following method.
· Log onto the server from your computer (see section 5.2).
· Open your server folder.
· Find the GeneScan file of the gel you want to look at.
· Transfer the GeneScan file from your server folder to your computer's hard drive.
· In order to call alleles in Genotyper, the GeneScan file must be imported into the program. The following procedure can be used to import the file.
· Select "Import GeneScan File" from the File menu (QK = OA I).
· Find the GeneScan file for the gel you want to look at.
· Open this GeneScan file.
· Hit "Import All".
A category needs to be made for each marker so that the alleles can be properly called. The follow procedure explains how to make a category.
· Open a new Genotyper file by selecting "New" under the File menu (QK= OA N).
· Import the first gel with the new markers on it.
· Open the plot window under the Views menu
· Select the color you want to view.
· Label and filter the peaks (refer to section 6.2.3).
· Determine the size of the alleles for the marker. Write down all of the different allele sizes.
· Select "Add Category" under the Category menu (QK = OA L).
· In the window that appears, mark the following things
o In the "Name" box at the top type the name of the marker.
o Select the color of the marker in the pull down menu next to the box.
o In the box in the center of the window click on the circle next to "All peaks"
o In the size area, fill in the boxes with the range of the size of the alleles for that marker. The lowest size in the range should be 4-6 bases below the smallest allele for the marker and the upper size in the range should be 4-6 bases above the largest allele for the marker.
o In the "Mark with dye color" box, check the box next to the color for the marker.
o Click OK.
· Repeat this for each of the markers on the gel.
· Repeat this for size standard. In the size area, fill in the boxes with the size range of the standard.
· When a category has been made for each marker on the gel, select "Save" under the File menu.
To call alleles for each marker, you look at that marker individually. The following procedure is an easy way to do this.
· Open the plot window by selecting "Show Plot Window" under the Views menu (QK = OA Y).
· Select the color you want from the color buttons in the upper left hand corner of the window. This will correspond to the color of the marker you will be looking at.
· Highlight the marker you will be looking at in the category list by double clicking on it. (Be sure all other marker categories are not highlighted.)
·
Select "
o Check the box next to "The Size In bp"
o Check the box next to "Round to Integer"
o Hit "OK".
·
Select "
o Check the box next to "Remove labels from peaks whose height is less than 32 % of the highest peak in a category's range".
§ The 32% can be changed to adjust for smaller peaks that may be missed.
o Check the box next to "Remove label from peaks proceeded by higher labeled park with in 0 to 1.6 bp" (these numbers can be changed).
§ Check the box next to "Higher by at least 5%"
o Check the box next to "Remove labels from peaks followed by higher, labeled peak with in 0 to 3 bp" (these numbers can be changed).
o Click on "OK".
· Zoom out to full range by selecting "Zoom Out (full range)" under "Zoom" in the Views menu (QK = OA H).
· Select the area where the marker system is by holding down the button on the mouse and drawing a box around the area.
· Now zoom in on that selected region by selecting "Zoom In (selected range)" under "Zoom" in the Views menu (QK = OA R). This will allow you to easily look at the specific marker.
· You can also zoom in and out in small portions to make the picture clearer. To zoom in and out, use the "Zoom In" and "Zoom Out" options described above (QK = OA + and OA -, respectively).
· Now that you can clearly see all the possible alleles for the marker, you need to check each lane individually to be sure all the appropriate peaks for the alleles were labeled. If a peak wasn't labeled and it should be, click on the peak to label it. If a peak was labeled and it shouldn't be, click on the peak to erase the label. Also make any note of peaks that could not be called because of standard mistakes, shifted lanes, and any rounding that occurred.
· When all the lanes have been checked, you can continue to the next marker selecting the color to be used and the marker that you will be looking at. (Be sure to unselect a marker when you are finished with it.)
· Continue checking all markers on the gel until you are finished.
· Be sure to save this new Genotyper file by selecting "Save as" under the File menu. Put it in your server folder and hit "Save as".
After all the alleles are called for all of the markers, they can be put into a table and then exported to an excel file using the following procedure.
· While holding the "SHIFT" key, select the colors of the markers that are present in the gel.
· Next, highlight the markers in the category window that are used on the gel.
· Select "Set Up Table" under the Table menu. Be sure all of the following are marked.
· In the "Contents per Row" line, click on the circle next to "Sample".
o Check the box next to "Sample Info".
§ Click the "Options" key.
§ When the window comes up asking for number of columns, type 1.
§ Click on "OK".
o Check the box next to "sample comment".
§ Click the "Options" key.
§ When the window comes up asking for number of columns, type 1.
§ Click on "OK".
o Check the box next to "Labels".
o Click the "Options" key.
§ In the "Number Of Peaks Per Category" box type 2.
§ In the "Labels Per Peak" box type 1.
§ Under the "Arrange column so that" heading, click the circle next to "Labels from same peak are next to each other".
§ Check the box next to "If only 1 labeled peak in category, then duplicate the label".
§ Check the box next to "If category has no labeled peaks".
§ Click the circle next to "Put this text in cells" and type 0 in the box.
§ Check the box next to "If some labeled cells are empty, put this text in empty cells:" and type 0 in the box.
§ Click on "OK".
o Check the display at the bottom of the window to be sure it displays what you want to see.
o Click "OK".
· Select "Append to table" under the Table menu.
· Now, select "Export Table" under the Table menu. Name the file to signify what is being displayed in the file (i.e. family and marker present). Hit the "Save" key.
· Save the Genotyper file with the new table included.
After the tables have been exported to excel files, they must be corrected for rounding errors and sorted in the appropriate order by performing the following steps.
· Open the excel file you want to work with.
· All rounding errors need to be corrected using the following steps
o Highlight the column with information for one marker.
o Select "Replace" from the Edit menu (QK = OA H).
o In the "Find What" box type the number you want to find.
o In the "Replace With" box, type the number you want to replace it with.
o Hit the "Replace All" key.
· Continue to the next number to be replaced until all are changed to the appropriate numbers.
· When the first marker is finished, continue to the next marker.
· When all of the markers are finished, highlight all of the columns with information.
· Select "Sort" from the Data menu.
· In the window that appears, signify how you want the information sorted.
· Hit the "OK" button.
· Save the excel file by selecting "Save" under the File menu.
· Be sure the name is right and the file is in the right folder
· Click "Save".
· It will ask you if you want to replace the file that exists. Hit the "Replace" key.
7.1: Overview
7.2: Operation
7.3: Startup Procedure
7.4: Shutdown Procedure
The Hydra 96 is an automated liquid handling device for use with 96 and 384 (with adaptor) well microtiter plates (PCR plates). The primary use for this machine is to multiplex and mix PCR products in preparation for running on the 377. It can also be used to setup PCR reactions and perform dilutions, among other things. The machine works by using 96 titanium tipped 100ul glass syringes. It has an integrated automatic wash unit to clean the syringes after each use.
· Check the wash fluid reservoirs located under the bench. There are three reservoirs; one bleach solution, one deionized water and one waste.
· Choose the correct file (program) to use. See the "Programs" section for details.
· Aspirate versus Dispense: With aspirate you pick up multiple times and dump once. With dispense you pickup once and dump multiple times.
Note: This procedure assumes that the Hydra was properly shut down using the "Shutdown Procedure" below. Make sure that the "DI water for rinse" tray is still on the tray table in the Hydra.
1. Turn the Hydra ON using the power switch on the back of the machine. The display should read "Empty!", but there will still be water in the syringes.
2. Press the "SET/RESET" button once and the display will read "File 1"
3. Press the "SET/RESET" button until the display reads "Empty!"
4. Press the "EMPTY" button to aspirate the water out of the syringes. The display should now read "A 0.0"
5. Remove the "DI water for rinse" tray. The Hydra is now ready for use.
Note: In order to protect the syringes, they MUST be stored in DI water when the Hydra is not in use. Use the following procedure when you are finished using the Hydra.
1. Place the tray marked "DI water for rinse" onto the tray table, making sure that it is centered under the syringes. Also make sure that the tray is 1/2 full of DI water.
2. Press the "SET/RESET" button until the display reads "File x" (x = any number).
3. Press the "UP" or "DOWN" button until the display reads "File 16"
4. Press the "SET/RESET" until the display reads "A 0.0"
5. Press the "FILL" button and watch the display as the syringes fill with water
6. When the syringes contain 30-40 μl of water, press the "Emergency Stop" button to shut the Hydra down. Leave the "DI water for rinse" tray in the Hydra.
7. Turn the "Power" switch on the rear of the Hydra to "OFF" and disengage the "Emergency Stop" button by turning it clockwise (it should pop back up).
Table of Contents
8.1: 10x TBE
8.2: 1x Gel Buffer
8.2: Polyacrylamide Gel Solution
8.3: Loading Mix
· 108 g Tris Base
· 55.0 g Boric Acid
· 8.3 g EDTA (disodium)
· Deionized distilled H2O to 1 liter.
Verify that pH is 8.3 plus/minus 0.2
Filter TBE that will be used in gel preparation. Discard any TBE that precipitates, do not autoclave.
· 150 ml 10x TBE
· 1350 ml ddH2O
· 14.4 g Urea (at least electrophoresis grade)
· 4.8 ml 40%; (19:1) Acrylamide/Bisacrylamide
· 20 ml ddH2O
· 1 g Amberlite Resin
Mix all ingredients in a beaker and stir until solution is clear (10 minutes minimum). During this time the resin will grab ions present in the solution. Filter solution through a 0.2 micron filter, and degas for 5 minutes by leaving vacuum on. Bring to 36 ml with ddH2O, store in dark until ready to pour gel. Solution is stable for 48 hours.
· 250 μl deionized formamide
· 25 μl GeneScan-500 size standard (ROX)
· 25 μl Loading Buffer
Can use less ROX than ABI recommends. Some customers use 0.3 μl of ROX per sample instead of 0.5 μl). Increase amount of formamide as needed to total volume wanted. Gently vortex to mix and use immediately.
The information for this section was adapted from a protocol
developed by Shawn R. Carlson in Brian Diers' lab at the