Roe Lab Gel Protocols

Version 3-9-98

The following is a list of reagents and protocols used daily to ensure that nine 377XL Applied Biosystems DNA Sequencing machines are successfully loaded 2-4 times per day. Follow these instructions meticulously. If you have any questions, see Lisa or Heather.

Part I: Solutions

A. Making Gel Mixes From Scratch

Please be sure to log the solutions in the log book.

**Note: Polyacrylamide gel mix contains neurotoxic materials. Always wear eye protection and gloves while handling.

1) 5.3% Long Ranger Polyacrylamide Gel Mix

a) Add 360 g of urea to a 1L beaker.
b) Add 400 mls of ddwater and dissolve. (*may require heat)
c) Add 100mls of 10XTBE
d) Bring to volume of 900mls with ddwater in a 1L graduated cylinder.
e) Parafilm and invert to mix.
f) Remove 6mls of the homogenous solution with a seriological pipet.
g) Add 106mls of FMC 50% Long Ranger stock solution.
h) Parafilm and invert to mix.
i) Place a stir bar into the 1L Nalgene filtering apparatus and place on a stir plate.
j) Pour gel mix into a 1L Nalgene filter unit.
k) Filter, stir, and degas gel mix for 20 minutes.
l) Remove filtering piece, and place lid on bottle. Foil and label the bottle. (sol'n, date, bottle #)
m) To pour a gel, aliquot 100mls of 5.3% solution into a 250ml beaker and add a stir bar.
n) Add polymerization reagents (650ul of 10% APS and 50ul of Temed). Stir for 5 seconds and pour the gel.

2) 5.0% Long Ranger Polyacrylamide Gel Mix

a) Add 360 g of urea into a 1L beaker.
b) Add 400mls of ddwater and dissolve. (*may require heat)
c) Add 100mls of 10XTBE.
d) Bring to volume of 900mls with ddwater in a 1L graduated cylinder.
e) Parafilm and invert.
f) Add 100mls of FMC 50% Long Ranger stock solution.
g) Parafilm and invert.
h) Place a stir bar into the 1L Nalgene filtering apparatus and place on a stir plate.
i) Pour gel mix into a 1L Nalgene filter unit.
j) Filter, stir, and degas gel mix for 20 minutes.
k) Remove filtering piece and place lid on bottle. Foil and label the bottle. (sol'n, date, bottle #)
l) To pour the gel, aliquot 50mls of 5.0% solution into a 250mls beaker and add a stir bar. Add polymerization reagents (325ul of 10% APS and 25uls of Temed). Stir for 5 seconds and pour the gel.

B. Making Other Solutions

1) 10X TBE (ABI) Buffer--MADE DAILY

Add the following reagents to a 2000 ml beaker:

216g TRIS
110g Boric Acid
16.6g EDTA

Fill beaker to ~1800 ml with ddwater, stir to dissolve, and bring to volume in a 2L graduated cylinder. Divide into two clean 1L buffer bottles.

Label and date each bottle with your appropriate colored tape.

Make sure there are 4-6 liters available upon your departure!!

2) 10% Ammonium Persulfate

Weigh 1.0g of APS into a 10 ml graduated cylinder and fill to 10 ml with ddH2O. Parafilm the top of the cylinder and shake until APS is completely dissolved. Pour the solution into the bottle marked 10% APS. Never make more than 10 ml at a time.

3) TEMED

TEMED is stored in the ABI refrigerator. Remove one bottle and write the date on the label. It is very important to parafilm the TEMED after it has been used.

4) Deionized Formamide

In a beaker, use 10g of Amberlite per 100 ml of formamide. Stir this solution for 1 hour to deionize. Filter the solution using the 150 ml Nalgene filtering system. Store in a dark bottle in the ABI refrigerator.

5) 25mM EDTA, 50 mg/ml Blue Dextran

Add 0.93g of EDTA to 90 ml water. Then, adjust the pH to 8.0. Bring the final volume to 100 ml. Next, add 50 mg Blue Dextran to a 1 ml EDTA solution.

Part II: Plates

A. Washing

*WEAR GLOVES*

This method is the easiest, most efficient, and most effective way to clean the plates. It is very important that the plates are cleaned thoroughly

every time or there will be problems when pouring gels. Remeber to keep the sets of plates together. The 377 plates are etched with numbers.

1) Place the plate in the sink so that the back plate is facing you. Remove the comb and place in the sink. (The back plate is the plain glass one and the front plate has the notch at the top.) Pry the back plate away from the front plate using a Sooner Scientific plastic wedge. Remove both spacers and place in sink. Place a Kimwipe onto the plate with the gel mix and pull up, releasing the gel. Discard into biohazardous waste bag.

2) Wash the front plate first. Turn it so that the etched lines at the top and bottom are facing away from you. These lines indicate the outside of the plate. Slant the plate in the sink by supporting it at the top with one hand. Using HOT water and a lot of pressure, rinse the small gel pieces off the plate. Scrub the surface of the plate using gloves.DO NOT USE A SPONGE OR ALCONOX TO CLEAN THE PLATES!!! While cleaning, be sure to clean the sides, top, and bottom along the edges. After one side is washed, turn the plate over and wash in the same manner. When the plate is completely clean, rinse it well with ddH2O and place it standing up in the trays in the fume hood, again with the etchings facing away from you. Rinse the plate with 95% EtOH to take off the water.

3) Now wash the back plate. Again, scrape the gel remaining on the plate into the biohazard bag. Place the plate in the sink slanted with the gel side facing you. Rinse the plate, especially the top, with hot tap water. Wash both sides of the plate thoroughly, keeping track of which side was on the inside (the gel side). Rinse with ddwater, place the plate in the fume hood with the inside facing you and rinse with ethanol.

4) Rinse both spacers and the comb with warm water. Place into dish drainer, then dry.

B. Pouring

1. Pouring Gels Using ABI Cassette Apparatus (5.3% Long Plates Only)

a) After the plates have dried, place the back plate onto the gel case with the inside facing up. It will fit into the grove and stop when it touches the metal prongs. Make sure it is secure.

b) Next, place the spacers onto the back plate, making sure that the spacer is flush with the edge of the plate.

c) Then, place the front plate directly on top of the back plate, with the spacers in between. Make sure the bottom of the plates are lined up with each other in the case.

d) Turn the clamps onto the plate in order to secure its position.

e) Attach the back support of the comb section. at the top of the gel. Slide the fixture in underneath the gel cassette until it stops against the top edge of the bottom plate.

f) Attach the bottom pouring fixture. First, using a syringe filled with air, blow air through the channel where the gel will enter to ensure that no polymerized gel is present. Then, hold the fixture against the bottom plates and gently, simultaneously turn the handles down on each side to lock the fixture into place.

g) Attach the small clear brace of the top fixture by sliding pegs under the pair of clamps located at the top of the gel. Insert the 48 well shark's tooth comb. Tighten the screws until the comb area is firm, but the comb is allowed in and out of the area. Keep the clamps open.

h) Have the comb and a clean syringe readily available.

i) Prepare gel mixture as stated on page 1.

j) Pull the swirled mixture into a 60 ml syringe and proceed to insert the syringe into the channel of the large fixture at the bottom of the plate.DON'T SUCK UP BUBBLES!

k) Gently push the solution out of the syringe into the plates. Knock on theplates gently to avoid the formation of bubbles.

l) After the gel solution has migrated up to the top of the plates, add the shark's tooth comb on the flat side.

m) Tighten the clamps onto the plates and the comb area. Immediately place a paper towel saturated with water over the bottom portion of the plates. Allow the gel to polymerize for 1.5 hours prior to use. Wrap and label the gels with date and bottle #, then place in the cabinet.

n) Be sure to thoroughly clean the gel case parts. Dry them off and place them in the appropriate areas.

2. Pouring Gels Using Clips (5.0% Short Plates Only)

a) Assemble the plates as with the cassette. Place the back plate with the inside facing upwards, then place the spacers on the plate as directed above. Place the notched plate on top of the back plate, making sure the plates are flush at the bottom.

b) Secure the plates with two clips equally -near the top of the plates.

c) Prepare the gel mixture as noted on page one and pour moving a beaker continously across the plate while knocking.

d) After the gel migrates via capillary action, disperse 3 additional sets of clips evenly on the plate. Place one clip in the middle of the comb area, not disturbing the comb placement.

e) Immediately place a paper towel saturated with water over the bottom portion of the plates. Allow the gel to polymerize for 1.5 hours prior to use. Wrap and label the gels with date and bottle #, then place in the cabinet.

3. Pouring Gels Using the Casters (Short and Long Plates)

a. Using the Sooner Scientific Caster (Short Gels)

1) Lay the back plate facing inward onto the casting device.

2) Place the appropriate spacers onto the back plate2

3) Place the bottom half of the top plate onto the top of the bottom plate. Rest the top plate on the casting device.

4) Prepare the gel solution by adding the specified amounts of reagents.

5) Pour the gel mix onto the bottom plate, while sliding the top plate until the ends of the plates are flush.

6) Clip the plate set using the above method and insert the comb. Remove plate set from the caster.

7) Immediately place a paper towel saturated with water over the bottom portion of the plates. Allow the gel to polymerize for 1.5 hours prior to use. Wrap and label the gels with date and bottle #, then place in the cabinet.

b. Using the Stretch Otter (Long Gels)

1) After the plates have dried, place the back plate onto the Otter with the inside of plate facing up.

2) Using a damp paper towel, wet both sides of the spacers and position them onto the back plate. Make sure that the spacers are flush with the bottom edge of the plate.

3) Place the bottom half of the front plate with the inside facing down onto the top half of the back plate. The top plate will rest on the otter and hang off of the front edge. The insides of the plate must be facing each other.

4) Prepare the gel solution as noted on page one.

5) Pour the gel mix onto the back plate while sliding the front plate onto the back plate. Continue pouring the gel mix and sliding the plates until the bottom edges of the front and back plates are flush.

6) Insert the specified comb into the gel. Clip the plate set using the tighter clips around the comb area and looser clips around the bottom of the plate set.

7) Remove the plate set set from the Otter.

8) Immediately place a paper towel saturated with water over the bottom portion of the plates. Allow the gel to polymerize for 1.5 hours prior to use. Wrap and label the gels with date and bottle #, then place in the cabinet.

c. Wrapping the Gels

1) Fold two paper towels in half.

2) Saturate with diluted 10XTBE

3) After polymerization is complete, place one paper towel on the front end and back end of the plate set. Fold the sides of the paper towel around the sides and end of the plate.

4) Use handi-wrap to seal the paper towel and secure with a rubber band.

Part III:Setting up and running the 377 ABI DNA Sequencer:

A. Preparing to Plate Check

  1. Unwrap the top and bottom of the gel, being careful not to bump the comb. Slowly remove comb, focusing attention on the edges of the formed well. If the comb is removed too rapidly the well will become deformed.
  2. Wash the gel to rinse dried gel off of the glass plate. Make sure all gel debris is washed from the edges of the glass.
  3. Remove excess gel from the beveled edge of the front plate using the square corner of a retired 48-well comb. Using a single tooth of the same comb, remove any smaller gel particles from the preformed well being careful not to touch the gel itself.
  4. Make sure the plate is free of del debris and urea, then rinse with double distilled water and place in the vent hood.
  5. Rinse the bottom 1/3 of the plate with 95% EtOH. DO NOT GET ETHANOL IN THE WELL OF THE GEL!!
  6. Remove the previously run gel from the ABI machine. Dump the old buffer out of the bottom buffer chamber, then replace it back on the machine.
  7. Restart the computer by pulling down Special and select Restart. Double click the Hard Drive icon, then double click the ABI prism 377 icon. Double click the ABI prism 377 collection icon.
  8. There must be at least 150 MB of disk space available before starting. If there is not, the oldest run file should be copied to a lab Jaz disk. After copying is complete, be sure the run file make it to the Jaz disk by double clicking on the Jaz icon and look for the file that was just copied. After double checking, eject the jaz disk by dragging it to the trash. Then drag the file that was copied onto the Jaz disk into the trash. Under Special select Empty Trash. If will ask for verification. (Usually throwing away one run file will free up enough disk space on the computer.
  9. If you had to free up some disk space, you will have to double click the ABI prism 377 collection icon again. Pull down Window and select Preferences. Move the mouse to the right and release on Default file names. Under sample sheet, run folder and run file type your initials. Under sample file, the box on the left should state <sample name>. The box in the middle should have nothing in it and the box on the right should state <none>.
  10. Pull down file name and select Sequence Sample Sheet defaults. Under Dye Set/Primer make sure it reads "DT {BD Set Any-Primer}". The Intrument File should read "bd_abi 2" or the ABI machine # you are loading. (This is the example for ABI 2).
  11. Pull down Sequence Sample Sheet defaults and select Sequence Run defaults. Under operator, type your initials.
    Make sure the following is correct:
    		Short gel with 64-well comb		Long gel with 64-well comb
    		WTR - 36 cm				WTR - 48 cm
    		PreRun Module - PR 36A-2400		PreRun Module - PR 36A-2400
    		Run Module - Run 36E-2400		Run Module - Run 48E-1200
    		Lanes - 64				Lanes - 64
    		Autoanalyze with - <none>		Autoanalyze with - <none>
    
    and then, if all is OK, click OK at the bottom of the page.
  12. Double click the Sample Sheet folder. Throw old sample sheets in the trash. Old sample sheets have 1) someone's initials, 2) date, and 3) time. For example: wm3/2/98 11.27 AM.
  13. Double click on Make ABI Sample Sheet folder. In order to use the Make ABI Sample Sheet_OU program, you must have the following information: the researcher's project code, the block name(s) and the direction of the reaction (forward or reverse).
    1. Type 3 to create a sample sheet for a 64-well comb. Press Enter.
    2. Type the two character code for the project. The characters can be two letters or a letter and a sigle digit number (aa or a1 for example). Press Enter.
    3. Type the two character code identifying the first 96-well plate you are loading. The characters must be a letter and a single digit number (b1 for example). Press Enter.
    4. Type the two character code identifying the second 96-well plate you are loading. The characters must also be a letter and a single digit number (b2 for example). Press Enter.
    5. Identify the reactions as forward or reverse by typing f1 for forward or r1 for reverse. Press Enter twice.
  14. The program will automatically configure the appropriate sample sheets. Select the appropriate sample sheet according to the order you are loading the machines by selecting Samplesheet64h_1 for the first machine (this will be for lanes 1-8 on the first 96-well plate). On the second machine select Samplesheet64h_2 (this will be for lanes 9-12 of the first plate and 1-4 of the second plate). On the third machine select Samplesheet 64h_3 (this will be for lanes 5-12 of the second plate).
  15. Check your sample sheet on each machine. The sample sheet will have a list of sample names that look like this: b1a01aa.f1 where the b1=the block name, a01=the actual well from the 96-well plate, aa=the project name and f1=forward direction. Each sample name on the first machine should begin with the first 96-well plate you are loading. Each sample name on the second machine should have a combination of the block name for the first plate and the second plate beginning each sample name and the third machine should have each sample name beginning with the block name of the second plate you are loading.
  16. While checking the sample sheet also make sure the Dye Set/Primer is {BD Set Any-Primer} and the Instrument File is bd_abi # (where # is the assigned ABI machine number).
  17. After you have checked to make sure everything is correct, under File select Save As. This is asking you how you want to save this information, just click on Save.
  18. Under File select New. Click on the Sequence Run icon.
  19. Check the loading parameters. Collection time should be 3.5 hours for a short run and 10 hours for a long run. The plate check module should read "plate check A". All other parameters should be the same as noted earlier in preferences. Click in the sample sheet box and release on the newly created sample sheet (with your initials, date, time).
  20. Place and secure your clean, dry gel into a clean, dry cassette. Make sure the scan zone of the gel is clean. Align the comb in the well and slowly begin to insert the teeth of the comb into the gel. Continue to insert the comb until the top of the wells create a semicircle above the glass. Once the comb has punctured the gel, it should not be removed.
  21. Put the upper buffer chamber on taking care not to bump the comb.
  22. Place the cassette into the machine. Secure by placing the palm of your hand in the center of the plate, gently pushing inward and rotate the four knobs to secure the cassette. Plug in the black connector for the upper buffer chamber to the machine.
  23. Shut the ABI door and begin to plate check the gel by selecting the Plate Check button. A scan window should appear with four colored lines: Red, Black, Green, and blue. These lines should be relatively straight. Always check the gel image under the Window menu.
  24. Your goal for a satisfactory plate check is a black screen! A single, sharp vertical line is a scratch on the glass plate and there is nothing you can do to correct that. However, if you have haze, there are several things you can try before you trash the gel:
    1. pull the cassette off then put if right back on
    2. put the cassette on another machine
    3. put the plate in another cassette (you may have to do this more than once)
    4. if any of these three things changes the gel image, it is not a bad gel, however, if you see no improvement you can:
    5. use a kim wipe with EtOH on the bottom of the plate again to try and clean it better
    6. last resort - wash another gel
  25. Cancel the "plate check" if the plate is satisfactory. You will be required to verify the cancellation.
  26. Make 1X TBE by filling a 2L graduated cylinder with 1800 ml of ddH2O and 200 ml of 10X TBE. Parafilm and invert to mix. Pour buffer into the upper buffer chamber until the buffer level is approximately 1cm from the top of the chamber. Fill the bottom buffer chamber with buffer until the level reaches the black plastic at the bottom of the cassette.
  27. Put the hot plate on and click on the PreRun button. (Allow the ABI machine to prerun for at least 15 minutes before loading the samples).

B. Preparing the DNA:

  1. Add 30 µl of loading dye and 100 µl of deionized formamide (found in fridge) into a 1.5 ml microcentrifuge tube, mix thoroughly. This is enough for one 96-well plate. You will need to multiply by the number of plates you are loading to have enough.
  2. Using a repeat pipetter, dispense 1 µl of the dye/formamide mix onto the side of each well. Cover the 96-well plate with an acetate sealer.
  3. Centrifuge the 96-well plate at 1500 x g for 30 seconds.
  4. Heat the samples at 100[ring]C on the top rack of the oven for 3-4 minutes. DO NOT HEAT LONGER THAN 4 MINUTES!
  5. Remove the samples from the oven and immediately vortex the samples thoroughly, then re-centrifuge at the same conditions listed above. The DNA can now be loaded.

C. Final Instructions before Loading:

  1. Under Window select Status. This will allow you to view that the machine is operating correctly. Pay close attention that the current is correct for the PreRun module (~1kV) and the time alloted for the PreRun is 1 hour.
  2. Open the door and you should get a message "Instrument Paused". Leave the door open. The gel will continue to heat but no electrophoresis will occur.
  3. Fill a 30cc syringe with buffer from the upper buffer chamber. Attach an 18 gauge needle to the syringe. Aim the needle toward the wells and begin to gently press on the plunger, be careful not to touch the comb. This will remove bubbles, small gel particles and urea from the wells. Be careful when raising the syringe back out of the buffer because there will still be a stream of buffer coming out of the syringe. A little back pressure on the plunger will stop this stream. Now you are ready to begin loading.

D. Loading:

  1. Carefully and gently expel any ethanol that is remaining in the 8-channel pipet. To rinse the ethanol from the 8-channel pipet, push up on the bar and take in buffer from the upper buffer chamber, then push down to expel buffer. Make sure each syringe expels buffer. Rinsing the syringe is repeated each time samples are loaded onto the gel.
  2. Place the 8 syringes in the bottom of each well in column 1 of the V-bottom 96-well microtiter plate and slowly lift upward on the bar loading the DNA from each well into each syringe (this takes some practice).
  3. Place the first needle into the first well of the comb, the other needles should line up with the other wells (the needles should line up on every fourth well).
  4. Carefully and slowly begin to pipet the samples into each appropriate well. If bubbles show up in the well, gently lift the entire 8-channel pipet up until the bubbles float out of the well, then continue.
  5. Rinse and place the 8-channel in column 2 of the plate, load the DNA into the syringe.
  6. Skip 3 wells to the right of the eighth sample pipetted, and place the first needle in that well (it will be well 33), letting the other needles line up with their corresponding wells. Pipet the DNA into the wells.
  7. Rinse, close door, and allow the samples to pre-run for at least 3 minutes. (When you open the door for the next step, the wells that were just loaded should look empty with a dark blue line at the bottom of the well).
  8. Open the door and load column 3 starting with the second well in the comb, rinse, load column 4 in well 34, rinse, close door and allow to pre-run for at least 3 minutes.
  9. Open the door and load column 5 starting with the third well, rinse, load column 6 in well 35, rinse, close door and allow to pre-run for at least 3 minutes.
  10. Open the door and load column 7 starting with the fourth well, rinse, load column 8 in well 36, rinse, close door and allow to pre-run for at least 3 minutes.
  11. Cancel the PreRun. You will have to verify the cancellation. Click on the Run button. A screen will come up that says "gel file". Name the gel with the name or initials of the person whose samples you are loading followed by the machine number, the word gel and the date.
    For example: pan6gel3-2. After you have named the gel file, click on save.
  12. The run has started. To see the gel image, under Window select Gel Image.
    **Note: You would do the exact same loading procedure for each machine when loading three machines at a time. Only on the second machine, the first sample loaded into the first well would be column 9 of the first plate and column 10 would go in well 33 etc. For the third machine, the first sample loaded would be column 5 of the second plate and column 6 would go in well 33 etc.

E. Final Paperwork:

  1. Get a log sheet and fill it out.
  2. Make sure you put the tape from the gel on the general comments section.
  3. The results file is named R followed by the number of the month the day of the month followed by a letter corresponding to the assigned run on the ABI machine and the last two digits of the year. For example, if you loaded the first run of the day on January 2, 1998 on ABI 5, you would name the results file: R01.02i.98

F. Cleaning Up After a 377XL Run

The next part of loading the gels is cleaning the area so that others will be able to load free of hassle.

  1. Throw away your microtiter plate and plate sealer if you no longer need it.

  2. Clean up any trash that might be around the ABI machine.

  3. Rinse 8 channel with ddH2O and Ethanol. Leave Ethanol in the syringes and return to its case.

  4. Place the 2L cylinder used for buffer on the cart in the ABI aisle.

Part IV: Data Analysis after a 377 Run Using the PowerMacs

To use the Power Macs for data analysis you must do the

following:

1. On the ABI Macintosh:

a. Open the Run file, then locate the folder containing your gel file. Highlight the log and collection files and drag to the trash. Empty the trash. The only item remaining in the folder should be your gel file.

b. Pull down Apple, select Chooser. Then do the following:

1) Click on Appleshare in Chem zone and choose PowerMac 1-4, click on OK.

2) Type in name (abi) and password (roelab), click on OK. Close the chooser window.

3) The appropriate PowerMac icon should appear in the upper right hand corner of the desktop.

4) Double click on the icon to open the power mac and copy the folder containing the gel file into a folder that is labelled with your initals. When copying is complete, close all the windows and restart the computer.

2. On the Power Mac:

a. To perform data analysis, open your initialed folder and double click on the gel file. The ABI analysis software will automatically begin to track and extract the gel file. This will take several minutes.

b. Samples need to be retracked because the current software is unable to auto track accurately. Pull down File and select Save.

c. Discard the results file from the initial tracking.

d. With the gel file open, pull down Sample and select Generate New Sample files. This will reanalyze your gel with the proper tracking and create another results file.

e. Name the results file R followed by the number of the month . the day of the month followed by a letter corresponding to the assigned run on the ABI machine . and the last two digits of the year. For example, if you loaded the first run of the day on January 2, 1997 on ABI 5, you would name the results file: R01.02i.97

f. When extracting and tracking is complete, the computer will display a Sample File Queue. You must add your files to this queue by:

1. Click on Add Files, select the newly created results file.

2. Click the box that says "Add All". The files will transfer from the top window into the bottom window.

3. Click Done

g. Highlight all of the samples in the sample queue (press the shift key and drag down the list)

1. Set basecaller to Adaptive.

2. Click Custom

3. Change DyePrimer to DyeTerminator{Any Primer} for terminators.

4. Change instrument file to the ABI used in your run.

5. Click OK.

6. Click Start in the sample queue. Analysis will be more rapid than on the ABIs.

h. Once initial tracking is complete, the trace file data will be displayed. Raw data, EPT, file information and sequence can be accessed by clicking the small boxes in the bottom left corner of the data window. It is known that this version of the software does not accurately report the EPT data, therefore, it is not necessary to record these numbers. The base spacing, however, is correct and should always be recorded.

k. Before copying to HD669, delete the .Seq files from the results file.

1. Pull down Apple, select Chooser.

2. Click on NSFshare, then double click on HD669. Click OK. HD669 icon will now appear on the upper right corner of the desktop.

3. Copy ONLY the results folder containing the trace files to the abi folder on HD669.

f. When you are satisfied that your own data has been copied over and tracked correctly, it is your job to discard the folder containing the gel file and trace file from the powermac. The hard disk can only accomodate two days worth of data so please analyze as soon as possible and trash your results files that are not in use.

C. ABI Nomenclature...6-3-97

ABI 1:first run = a ABI 6:first run = j

second run = b second run = n

third run = e third run = r

fourth run = f fourth run = v

ABI 2:first run = y ABI 7:first run = k

second run = z second run = o

third run = aa third run = s

fourth run= bb fourth run = w

ABI 3:first run = c ABI 8:first run = l

second run = g second run = p

third run = dd third run = t

fourth run = ee fourth run = x

ABI 4: first run = d ABI9: first run = cc

second run = h second run = hh

third run = ff third run = ii

fourth run = gg fourth run = jj

ABI 5:first run = i

second run = m

third run = q

fourth run = u

Part V: General Maintenance

This is a list of duties that must be completed either daily, weekly, or monthly. These tasks help keep the entire lab clean and running smoothly. As undergrads, it is our job to help out in this area as much as possible. Most of these jobs take little time to do, but they make a world of difference when done on a regular basis.

A. DAILY TASKS

1) Glassware

All glassware used for making solutions or pouring gels should be cleaned and put away. It is your responsibility to clean the beakers used for pouring gels the day before. Wash out the glassware with hot tap water and rinse very well with dH2O. Put the glassware in the dish drainer next to the sink and put it away when dry. .

2) Stainless Steel Counter and Black Countertops

Spray the countertop with water and scrape all the gel off with the

scraper. With the squeegie, collect all gel mix particles into a biohazardous waste bag. Wipe with sponge and water until clean.

3) Reagent bottles

The APS and TEMED bottles tend to get dried gel mix on them while gels are being poured. Wipe these off every day. Make sure the other bottles are clean and wipe them off if they are not. Make sure the TEMED is parafilmed.

5) Pipets

The pipets used to measure the APS and TEMED also get gel mix on them. Always wipe these off after pouring gels with a damp paper towel.

6) Ethanol and ddH2O Carvoys

These need to be checked daily; especially on Friday for the weekend people. Get the water from the still at the big sink behind the weigh station. To fill the EtOH, take the carvoy on a cart down to the stock room. Using a funnel, pump the EtOH from the barrel until the carvoy is full. Jim in the stockroom will have you fill out the necessary paperwork.

7) Supplies

Always check to make sure we have all the supplies we need. Things like kimwipes, sharpies, boric acid, glassware, stir bars, clips, polyethylene bottles, latex and green gloves, etc. can be retrieved from the stockroom.

Other things like filters, urea, polyacrylamide, loading tips, combs, TRIS, etc. must be ordered so if you notice that these items are less than half, NOTIFY LISA OR HEATHER ASAP! Do not wait until these items are gone to tell someone we need to order them.

8) Daily Solutions

Check the solutions sheet for any solutions that need to be made. Consult the solutions manual for the reagents needed. If you have any questions seek help immediately. After the solutions has been autoclaved and placed on the shelf in the cold room, mark the solution off of the sheet.

9) Autoclaving solutions for the lab

Every morning, unload the autoclave from the previous evening. Autoclave all items on the "to be autoclaved" cart. Be sure to place media on the ready made shelf in the cold room.

10) Fill yellow pipet boxes

Check table in the main hallway for empty yellow tip boxes. Using gloves, fill the tup boxes, mark with autoclave tape and place on the "to be autoclaved" cart. After run is complete, place filled tip boxes in appropriately marked area.

11) Walk ABI aisle

Twice a day, walk the ABI aisle and pick up trash, cylinders, collect syringes, and empty tip boxes.

12) Cleaning Loading Tips

Fill empty loading tip boxes with used tips. Clean with ddwater, and dry in oven.

B. WEEKLY DUTIES

These tasks will probably be assigned to different people, but it is important that everyone know how to do these tasks.

1) Sinks

Clean the gel out of the sink and the drain. Pull up the mats and rinse them off. Scrub the sink with 409 and wipe off the bottles if necessary. Open the doors under the sink and unscrew the trap and empty it into the bucket. Screw the trap back on tightly. Put the sink area back in order.

2) ABI Aisle-endust electronics

Clean off the monitors and keyboards with Electronic endust and duster.

Wipe off the counters around the machines with 409 and a sponge. Check the supplies of loading tips.

3) Fumehoods

Clean any trash out of the fumehoods. Empty the ethanol out of the trays disposing of it in the ventilation hood in the organic solvents waste hood. Wipe down the surface of the hood with a sponge. Put everything back in order. It is also necessary to clean the balance and restock chemicals in the hazardous chemicals hood. In the organic waste hood, the EtOH must be siphoned into the appropriate waste container.

4) Cabinets

Wet the cabinets underneath the stainless steel counter and the black top counter and scrape off the gel. Proceed to wash over with water and to spray with 409 and wipe until clean. Periodically, open and clena the rim of the cabinets.

5) Refilling the Water Reservoir on the 377's

The water reservoir is located in a compartment on the right side of the instrument. Visually check the water level weekly and refill the water reservoir when it is between one-third and one-half full. Empty the reservoir container in the appropriate waste container and refill with the diluted antifreeze solution. Follow the below instructions.

a) Make sure the pump is not running.

*IMPORTANT* Do not remove the water reservoir while a run is in progress!

b) Open the water reservoir compartment on the right side of the instrument by pulling gently on the door.

c) Unscrew the plastic reservoir and remove it by pulling downward. Place a paper towel under the tubes connecting the reservoir to the pump to catch any drips.

d) Fill the reservoir with the diluted antifreeze solution to 3/4 capacity.

e) Replace the reservoir, being sure to insert the tubes that connect it to the pump before you screw it into place.

f) Close the compartment door.

7) Disposal of Biohazardous Waste

Disposing of biohazardous materials occurs two to three times per week. Collect all of the biohazardous waste flasks and bags. Make sure every liquid container is only 1/2 full and is covered with foil. Insert an odo-clave tablet. Make sure the bags are doubled bagged and taped shut. Autoclave materials in a bucket for 30 minutes. Promptly dispose of all of the bags in the trash, and dump the media out of the flasks. Wash all lab stock glassware and return other flasks to the appropriate persons. Restock autoclave cart.

8) Cold Room/Cyler Room

Using an Amphyl(tm) soaked rag, wash off the counter tops of the cold room.

Check the ready made solutions for used media and dispose.

Wipe off the counters that the cyclers sit on and pick up any trash.

Inform Heather of any contamination.

9) Cleaning the 377 Gel Cases

Take out all of the gel cases and run HOT water over them.

Turn the gel case knobs while under the water--attempting to loosen any dried

gel mixture. Dry off and replace in the cabinet.

10) Collect Lab Stock Glassware

Once a week, all of the lab stock glassware should be rounded up from around the lab and placed back in the glassware cabinet.

11) pH stations

The two pH stations must be wiped down and cleaned once per week. They are located in the main lab-next to the chemical stand and in the third lab-back left fume hood. The area should be washed down and the solutions should be check. Also, there should be a designated area for trash and spills.

12) Cleaning the Autoclave

While the autoclave is off, pour bleach (about 1/2 cup) into the trap.

13) Cleaning the refrigerator

Every Wednesday afternoon, the refrigerator in the break room should be cleaned out.

C) MONTHLY DUTIES

These duties are performed every Thursday of the month.

1) Acetic Acid Treatment of Plates

Wash plates as usual but do not rinse with ddH2O or EtOH. Place the plate in the rack in the fumehood, shut the sash partially and make sure you are wearing a lab coat, gloves, and protective eyewear. Rinse the plate with acetic acid and then with ddH2O. Bring the plate back to the sink and wash again, rinse with ddH2O and EtOH. Most of the time it takes two days to treat all the plates with acetic acid.

2) Freezers

In order to keep everyone's boxes in their proper place, it is best to clean the freezers shelf by shelf. Before you start, though, ask lab members which containers need to be kept frozen and put them in another freezer. Clear off the shelf and break off the ice with a hammer or screwdriver until all the ice is gone. Let the ice fall into a plastic bin. Always be wary of the coils for puncturing one will release freon and destroy the freezer. The -70 degree freezer is different in that it does not have shelves. Most of the items in this freezer need to be kept frozen. Remove the contents of the freezer and chip the ice out. The freezers are numbers and 2 of the -20 degree should be done each month.

3) Chemical Stand, Fume hoods and pH stations

Take all the bottles off the weigh stand and wipe them clean. Wipe off the top of the balance and take off the plastic cover and clean it off. Remove the paper and replace it. Put the chemicals back on the chemical stand in an orderly fashion. See weekly duties for description of fume hoods and pH stations.

4) Waterbaths

Clean out the waterbaths and add water to fill approximately 1/4 full.

Add 10 drops of algecide.

5) Hazardous Waste

Log any hazardous wastes onto the hazardous waste sheet--See Lisa-


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Bruce Roe, broe@ou.edu