BaseStationä Protocol
Preparation of PCR Plate:
For 96 Well Plate (Procedure can be modified):
For 30 cycle PCRs: Dilute your PCR product with H2O before proceeding. To know the correct dilution you will need to do a test run and dilute the PCR product in a series from 1:2 up to 1:20.
For 26 cycle PCRs: Follow the steps below without doing dilutions on your PCR product.
1. Prepare a master mix in 1.5 mL tube
a. 220 ul autoclaved Nanopure H2O
b. 155.2 ul formamide
c. 51.7 ul Dextran (loading dye)
d. 12.4 ul Ladder size standard Genescan (Rox 400, Rox 500, or a combination can be used depending on size range of markers)
2. Mix the master mix by pipetting up and down.
3. Transfer 1 ul of PCR product (undiluted or diluted depending on PCR cycles – see above) into another plate
4. Add 4 ul of master mix to 1 ul of PCR reaction
5. Spin down.
6. Heat for 3.5 minutes
7. Put on ice until ready to load onto gel.
Glass Plates and Assembly:
1. Using Kimwipes, clean glass plates with Nanopure H20, then with Ethanol.
2. Make sure plates and especially the wells are completely dry. Also make sure that the wells don’t have any gel fragments. If wells are not clean and dry, use tygon tubing connected to air to blow the water out of the well area.
3. Place bottom plate in MJ casting stand (making sure it’s flush with anode backstop). The arrows on the lower plate MUST POINT UP!
4. Lay the Mylar spacer on top of the bottom plate (align it with 4 corners of plate).
Note: Mylar spacers can be reused if rinsed off after use. Each spacer costs $2, so as long as the spacer is not torn or kinked, we should use it.
5. Place plate with wells on top of the spacer and make sure BOTH plates are flush against rear & far right backstops. (Loading well end of top plate will be opposite of clamps).
6. Clamp the glass plate and spacer together with binder clips before clamping down acrylamide injecting seal.
Gel Pouring:
7a. Ensure that the o-ring on the acrylamide injecting seal is properly seated & the ends of the o-ring are flush to one another.
CORRECT INCORRECT!
7b. Clamp down acrylamide injecting seal to buffer chamber (making certain it covers the slot).
· Note: Safety Goggles and Gloves must be worn when handling liquid acrylamide.
7c. Prep comb for insertion. Make sure screws are backed up into bracket so that the comb may be fully seated into the wells formed by the top plate.
CORRECT INCORRECT!
2. Add photoinhibitor to acrylamide. Amount to be added shown in chart below. If you are going to be running a lot of gels within 1 week you can add 1 ml of photoinitator to 49mL of acrylamide solution.
Go to GVL website for instructions on how to make your own acrylamide solution.
|
Plate Size |
% Acrylamide |
Acrylamide Stock |
Urea |
10X Buffer |
Total Volume |
Pouring Volume |
Photoinitiator Volume |
|
20cm |
5.5% |
5.5ml |
18g |
5ml |
49ml |
15ml |
300ul |
|
30cm |
5.0% |
5.0ml |
18g |
5ml |
49ml |
20ml |
400ul |
|
40cm |
4.75% |
4.75ml |
18g |
5ml |
49ml |
25ml |
500ul |
With a 25ml disposable syringe slowly draw up acrylamide, being careful not to draw up bubbles.
9. Attach syringe to anode seal and inject the acrylamide slowly until a front appears. Increase pressure to get an even front and constant flow until acrylamide reaches the well area on the cathode end and flows up through the all of the wells. Keep in mind that one wishes to have the acrylamide front moving fast enough to prevent the formation of bubbles, but not so forceful that bubbles in the buffer chamber are forced between the plates.
10. Insert the comb and make sure comb is flush with the bottom plate.
11. Unclamp anode seal and place syringe and anode seal in a waste bucket until you can clean the unpolymerized gel mix from the syringe and seal. Use the vacuum to clean the remaining unpolymerized gel mix from the anode seal.
Gel Polymerization:
12. Pre-run the UV light box for 5 minutes before putting in your gel. Remove the glass/gel assembly from the casting stand and transfer to the UV light box. Open the lid and place the assembly carefully in the center of the light box. Wipe of any unpolymerized acrylamide on casting stand with a kimwipe and put into UV box with the gel. The kimwipe can be thrown away after the acrylamide has polymerized.
13. The timer is programmed to expose the gel matrix to 5 minutes of UV light and shuts off automatically.
14. Close the lid and press Start on the timer (NO GLOVES).
15. Turn the gel around 180˚. Close the lid and press Start for a second run. To ensure that the gel is fully polymerized, the glass/gel assembly may be turned 180˚ again and a 3rd run may be performed. After polymerization, remove excess polymerized acrylamide from the UV box with nanopure/DI H2O using a paper towel.
While the UV box is running, turn on the 40˚C water bath. Make sure the reservoir has water in it!
16. Place the glass/gel assembly on the casting stand and remove binder clips.
17. With a 500ml squirt bottle and comb still inserted, rinse out cathode chamber with Nanopure/Deionized H20 to remove excess acrylamide. The H2O may be squirted directly onto the comb in the cathode chamber. Remove all water/acrylamide mix with the vacuum. Clean the region of top plate where BaseStation laser will by scanning once with nanopure H2O and then with ethanol using kimwipes.
BaseStation Protocol:
Change Settings: fully automated run to manual run or from manual run to fully automated run This only needs to be done when using a BaseStation that has the ability to auto-run gels!
1. Turn on the BaseStation and open up the BaseStation software on computer.
a. Select “Advanced” under “File” menu.
b. Password is “candide”
c. An options menu should now appear on top Menu Bar.
d. Select “Factory” under “options” menu.
e. Change the number of lanes to 48 or 96 under Hardware, on the right half of the screen
Setting up a fully automated run:
2. Make sure the DI water bottle on the side of the BaseStation is at least half full. Flush lines 3 times into an empty tip box. Go to the “Autoloader” pull down menu and choose “Flush Lines”. This will allow you to check for any clogged needles and ensure that the autoloader is properly moving. If the autoloader does not move, restart BaseStation and software. Make sure that liquid is dripping from all the needles. You can do this step without your gel in, but make sure the red front clamp is down.
3. Place gel on thermal plate, guiding the gel along the side rail to the spring loaded backstop. Push the assembly back with the front fence, until you can tighten down the front fence with the alignment pins.
4. Plug anode and cathode electrodes into their respective chambers.
5. Check for correct optical filter cassette: either BIG DYE (for sequencing) or GENOTYPING (for 6Fam, Tet, Hex) or GenoVicNed (for 6Fam, Vic, Ned).
6. On the BaseStation software menu:
a. In the “Settings” window, choose the appropriate Settings File and run conditions for the appropriate plate size. Typically “Automated Genotyping”.
|
Plate size |
Pre-Run Voltage |
Pre-Run Time |
Injection Voltage |
Injection Time |
Run Voltage |
Run Scans |
|
20cm |
1900 |
3 min |
5000 |
60 sec |
1900 |
15000 |
|
30cm |
2800 |
3 min |
5000 |
60 sec |
2500 |
20000 |
|
40cm |
3000 |
3 min |
5000 |
60 sec |
3000 |
23000 |
|
Genotyping |
1900 |
2 min |
4000 |
30 sec (no ramp) |
2600 |
6000 |
b. Select “Sample Sheet”. Name your gel: today’s date and name or description. It must not be more than 14 characters in length. (Example: 000823jen1a).
i. Under “Properties” tab select either sequencing or genotyping. Select plate size.
ii. Under “Loading Pattern” tab you can manually select your own loading pattern or import it from a previous gel with the same sample loading pattern and sample descriptions. To do this click “Import Sample Sheet”, double click previous gel file and then select the .xmp file that contains your loading pattern and sample descriptions.
iii. Under “Sample descriptions” tab you can type in all your sample names by hand or if you imported in the previous step your sample descriptions will have been filled out under this tab.
iv. Select the ladder that you used under “Set gel size standard”.
v. Under “Set gel panels” select none.
vi. Save completed sample sheet.
An alternative way to make your sample sheet: Make sample sheet on Excel (blank sample sheet can be found on lab 6\basestation\samplesheet). Save as text delimited file. Insert your sample loading pattern and descriptions into the “Sample Sheet” menu. Refer to the BaseStation Manual for directions on how to select a loading pattern. Import your sample sheet text file and save sample sheet. You can also type in your sample descriptions without importing from excel in this step.
7. Make 150ml fresh 1X TBE and fill back chamber. [135mL Autoclaved nanopure H2O, 15mL of 10 X TBE]
8. Note: You have 15 minutes to begin prerun once removing the comb. Remove comb by rotating the knobs clockwise simultaneously.
9. Add 135 ml of Nanopure/Deionized H20 to cathode (front) chamber.
10. Clean top plate if needed.
11. Slide thermal plate back and lower the single red front clamp to lock gel into place (make sure the locator pins on the bottom side of thermal plate are properly aligned with the gel locators). If the locator pins are not aligned properly, BaseStation will not autofocus.
12. On the BaseStation software menu click Plate Check. Click Yes to Autofocus gel. After autofocus, click play to perform plate check. If scan region is dirty, clean scan region and plate check again. If the scan region contains 1 or 2 small peaks continue running gel. If there are high peaks all over scanning region, autofocus the gel again and perform plate check. If this fails, you can continue to run the gel (which may look bad) or make a new gel.
13. Press stop to quit plate check. Click Yes to Quick Check the autoloader.
a. For left alignment, under new settings, type 4700 into the up|down box and press enter.
b. Use a flashlight and check to see if the first needle is in the third well.
c. Press Next
d. For right alignment, under new settings, type 5000 into the up|down box and press enter.
e. Use the flashlight and check to see if the last needle is in the third to last well.
f. Press Next.
g. Press Cancel.
14. If you are unfamiliar with Quick Check, ask somebody who has run the BaseStation before. If Quick Check is not done properly, mass destruction of needles will occur!
15. Click “Begin Run,” insert diluted PCR plate into 96 well plastic tray and fill buffer chamber with 10 X TBE. (The A1 well of the diluted PCR plate should be on the closest to you on the left)
16. At this point, you can leave the BaseStation to run on its own or you can watch the prerun to make sure the voltage and current looks good.
17. Fill out a BaseStation log sheet.
18. Clean comb with DI H2O, removing any polymerized acrylamide. Use vacuum.
Example of good injection:
Notice that the current drops after a gradual increase.
Setting up a manual run:
1. Under Settings chose manual genotyping
2. Sample Sheet (Refer to step 6b in setting up a automated run)
3. Add 150ml of 1 X TBE to anode (back) chamber
4. Remove comb and rinse with nanopure H2O 3 or 4 times. Use syringe to draw up the H2O after rinse.
5. Add 135 mL of H2O in cathode (front) chamber
6. Slide the thermal plate back and clamp down the gel with the front red clamp, making sure that the locator pins on the bottom of the thermal plate are aligned.
7. Click Pre-run on software screen
8. Load 1ul of each sample.
This can be done with a standard pipet but is much quicker with an 8-channel pipet. When using the 8-channel option it is important to remember which samples will be going in which lanes. The 8-channel loads into every 3rd well so typically you will want to set up your PCR plate so that samples you want loaded next to each other go in rows of 3.
Example: If you want samples 1-10 to be next to each other on your gel you will put them into the PCR plate in the following wells: A1=1, A2=2, A3=3, B1=4, B2=5, B3=6, C1=7, C2=8, C3=9, D1=10, etc, etc.
The first 24 samples will be loaded on the left side of the gel.
a. Using the 8-channel pipet, draw up 1 ul of each sample from column 1 of your diluted PCR plate.
b. Place the tip of your A1 sample into the well marked “1”. Make sure all other tips are also inside wells and don’t let the tip come to the very bottom of the well. Slowly eject the samples into the wells trying not to release air bubbles at the end.
c. Once samples are ejected, rinse tips by pipeting the water in the cathode chamber up and down three times. Make sure that the tips don’t have any water remaining in them after the final rinse. This process will be repeated for the next two columns.
d. The second set of 8 will be loaded by placing your A2 sample into the well marked “2”.
e. The third set will be loaded by placing your A3 sample into the well marked “ 3”.
The second 24 samples will be loaded onto the right side of the gel.
f. When loading samples from column 4 make sure that you place the sample from well H4 into the well marked “1” on the left side.
g. Repeat process for samples in columns 5 and 6 using the wells marked “2” and “3”, respectively, as guides.
9. Click Plate check and click yes when prompted to autofocus.
10. After autofocus click play button to plate check. Click stop button to stop plate check
11. Click Inject
12. After injection add 15 ml of 10X TBE to front chamber and mix briefly.
13. Start collection.
Clean-up