TITLE: SANGER Sequencing EFGL
AUTHORS: EagleFish GeneticsLab

[DESCRIPTION]
This protocol describes how the Eagle Fish Genetics Lab (EFGL) prepares extracted DNA samples for the ABI 3500xL Genetic Analyzer, and how we run and collect data from this machine. This process is achieved by PCR amplification, ExoSAP-IT purification, E -Gel confirmation, Qubit quantification, BigDye sequencing, CleanSeq clean up, set up of the 3500xL Genetic Analyzer (3500), and finally the collection of results. This should result in easy-to-read nucleotide peaks that are exported as an ABI file. 


[STEPS]
SECTION: PCR Amplification of Target Sequence
1. Materials needed:  
• Forward and reverse primers
• Heat Seal
• 1.5mL vials 
• PCR master mix reagents/kits 
• DNA tray(s) 
• Unskirted PCR plate(s)
• Pipette and tip 
SECTION: SECTION 1 – PCR Purification with ExoSAP-IT
14. Materials needed:  
• ExoSAP-IT Express reagent (pre-aliquot strip tubes in -20 °C small freezer) 
• PCR product plate 
• 1 unskirted PCR plate 
• p10 multichannel pipette and 2 boxes of tips 
• Ice block (in -20 °C small freezer) 
SECTION: SECTION 2 – Quantify Purified PCR Products with Qubit
23. Materials needed: 
• Qubit dsDNA high sensitivity kit 
• Purified PCR products 
• p10 multichannel pipette and box of tips 
• 1 unskirted PCR plate 
• Ice block(s) 
SECTION: SECTION 3 – BigDye Sequencing Cycle
31. Note: Reach out to Wildlife lab to make sure 3500 machine is available for use, prior to step 3. It is ideal to 
do STEP 3-5 on the same day.  If sequencing within next 1-3 days, it is OK to stop at either the end of step 3 or step 4. 
SECTION: SECTION 4 – Post Cycle Sequencing Clean-up
47. **Note** 3500 machine does injections in columns of 3. If you have a partial tray that the sample 
size is not a factor of 3 columns, or a full column → Fill it with water.  
For example: 
• If you have 4 columns of samples, fill column 5 and 6 with water. 
• If you have 2 columns + 7 samples, add water to the 8th well in the 3rd column. 
• Each injection takes ~ 120 min

SECTION: SECTION 5 – 3500 Sequencing Run
68. Make sure both computer and 3500 are turned on.  
          a. If 3500 is off, and computer is on. The computer needs to be restarted.  
                    i. Log in under Administrator, password is Administrator 
          b. Follow the instructions (bottom left corner) on 3500 to sync 3500 and computer. 
                    i. Data collection is the software that you launch to start the run. So, donot  open the 
software on the computer until you see the green check mark. 
          c. If 3500 is on, but you’re experiencing issues with the 3500 data collection software, reboot 
the computer. HOWEVER! Please make sure whoever is using that computer has their GeneMapper results saved! Please check with the techs in the lab.
SECTION: SECTION 6 – Collect Results
82. Go to “Computer” → Select “Data (D:)” → Applied Biosystems → 3500 → Data → Carp  
SECTION: Appendix – Successful PCR recipes
87. TaKaRa LA Taq (Cat# RR002A) - 20 µL reactions 

SECTION: Appendix – Successful PCR cycling programs
90. ND1_ND2 and dloop
SECTION: PCR Amplification of Target Sequence
2. To note: As there are several PCR kits our lab uses for this section, below is a generic protocol for 
amplifying PCR products for Sanger sequencing. Refer to the Appendix for our working recipes/programs. 
SECTION: PCR Amplification of Target Sequence
3. Obtain sample list from lead biologist or data coordinator, and print tray maps if necessary. 
          a. If this is your first attempt, start small (ideally 8-12 samples – as Step 4 in Post cycle 
          sequencing clean-up can be challenging).  
SECTION: PCR Amplification of Target Sequence
4. Consult the lead biologist on the master mix recipe and cycling program to use. 
          a. Appendix section’s recipes and programs have been successful at the Eagle Fish Genetics Lab,
              but please double check if those are the right ones for your lab.  
          b. Depending on the species and PCR product size, the PCR kit(s) could vary and/or be used in 
              combination. 
SECTION: PCR Amplification of Target Sequence
5. Locate forward and reverse primers stock (usually at 100 µM concentration) located in -20 °C freezer and dilute each individually to a working concentration of 10 µM).  
          a. In a 1.5mL vial, add 20 µL of 100µM primer to 180 µL of water. 
              Repeat for the other primer. 
          b. Store the 100µM stock back in lab -20 °C freezer and keep 10µM working stock in lab fridge. 

SECTION: PCR Amplification of Target Sequence
6. Prep PCR master mix in a 1.5mL vial. All reagents and/or kits are in our lab mini -20 °C freezer 

SECTION: PCR Amplification of Target Sequence
7. In a new unskirted PCR plate, dispense master mix into each well. 
SECTION: PCR Amplification of Target Sequence
8. Using a multi-channel pipette, add DNA samples into each well.
SECTION: PCR Amplification of Target Sequence
9. Heat seal, gently vortex and quick spin    down.  

SECTION: PCR Amplification of Target Sequence
10. Load the unskirted plate and run the appropriate thermal cycler program. 
(In most cases, these PCR runtimes can range between 240 min or longer, so plan accordingly) 

SECTION: PCR Amplification of Target Sequence
12. Run 1% e-gel to confirm the successful amplification of PCR products. Follow e-gel protocol found in: 
S:\Eagle Fish Genetics Lab\LAB PROTOCOL BOOK\05 - DNA quantification\E-Gel 
or on protocols.io under "E-Gel Protocol EFGL"
          a. Save e-gel images in a project folder 
          b. If successful, and depending on the sample size of the project, consider using double comb gels if 
              possible (more samples and less time) 
SECTION: PCR Amplification of Target Sequence
13. Repeat steps 7 to 13 for the remaining samples for your project. 
          a. It is NOT recommended to PCR all samples at one go, especially for a large project and the   
              availability of 3500xL Genetic Analyzer is entirely dependent on the Wildlife lab’s schedule. 
              They have dibs. 
          b. Stagger the sections, if possible. 
SECTION: SECTION 1 – PCR Purification with ExoSAP-IT
21. (If not using immediately) 
Heat seal and store in -20 °C freezer. (Samples are still stable and usable after a month 
[tested in May 2022])

SECTION: SECTION 1 – PCR Purification with ExoSAP-IT
20. Samples are now PCR purified 
The unskirted plates are to be placed on ice blocks for downstream steps.  
SECTION: SECTION 1 – PCR Purification with ExoSAP-IT
19. Run ExoSAP-IT program on thermal cycler. Runs for about 5 min. 
          a. User name: General 
          b. General → Sanger → Exo sapit 
          c. The program is currently only on our labs machine 1’s and machine 10’s groups as the Eagle Fish    
              Genetics Lab
SECTION: SECTION 1 – PCR Purification with ExoSAP-IT
18. Heat seal, vortex gently, and quick spin    down 

SECTION: SECTION 1 – PCR Purification with ExoSAP-IT
17. Add 2 µL of ExoSAP-IT Express to each PCR sample (ExoSAP-IT it is quite viscous, pipette slowly). 

SECTION: SECTION 1 – PCR Purification with ExoSAP-IT
16. Aliquot 5 µL of PCR product into a new unskirted PCR plate. 

SECTION: SECTION 1 – PCR Purification with ExoSAP-IT
15. Vortex ExoSAP-IT Express reagent and keep on an ice block (ExoSAP-IT doesn’t freeze).
SECTION: SECTION 1 – PCR Purification with ExoSAP-IT
22. 
 

SECTION: SECTION 2 – Quantify Purified PCR Products with Qubit
24. 
 

SECTION: SECTION 2 – Quantify Purified PCR Products with Qubit
25. Note: Remember that you will end up needing to sequence forwards and reverses separately (1 µL of 
purified PCR product each), and that after Qubit there will only be 5 µL of purified PCR product left. If the concentration is too low to do a forward and reverse run, you will need to ExoSAP more PCR 
product, and qubit them again to make sure you have enough.  


SECTION: SECTION 2 – Quantify Purified PCR Products with Qubit
30. When proceeding to the next step, purified PCR products should stay on ice block. 
SECTION: SECTION 2 – Quantify Purified PCR Products with Qubit
29. Heat seal, and store the original purified PCR product plate back in -20 °C freezer, if not using. 
          a. If you need to go back to these samples, note that the diluted samples may drop in DNA 
               concentration. These are still viable for sequencing after a month in -20 °C freezer.
               (tested in May 2022) 
SECTION: SECTION 2 – Quantify Purified PCR Products with Qubit
28. In some cases, these purified PCR products are often higher than 60ng/µl. (Qubit machine will read: 
“Too high”.) 
          a. For mitochondrial sequences:  
                i. Recommended to try a 1:10 dilution prior to Qubit, most samples should stay within 
                   10-30 range after dilution. 
                ii. 1:10 dilution: Add 1 µL of purified PCR product in 9 µL of water in a new unskirted   
                    PCR plate. 
                iii. Keep diluted plate on ice block as well 
          b. If 1:10 dilution doesn’t fall within the required range, calculate out the actual DNA present, 
               and either change the dilution factor or proceed with undiluted purified PCR products 

SECTION: SECTION 2 – Quantify Purified PCR Products with Qubit
27. Obtain the average qubit scores for each sample. Depending on the length of your sequencing products, 
refer to the table above to determine the required amount of DNA. 
SECTION: SECTION 2 – Quantify Purified PCR Products with Qubit
26. If the purified PCR product was previously frozen, it is OK to quick spin   to thaw (~5 s), gently vortex, and quick spin   down again. Remember to place plate back on ice block immediately after. 

SECTION: SECTION 3 – BigDye Sequencing Cycle
32. 
 

SECTION: SECTION 3 – BigDye Sequencing Cycle
33. Materials needed: 
• Forward and reverse primer (10µM concentration) 
• BigDye and BigDye Terminator v3.1 5X sequencing buffer 
• 2 of 1.5mL vials to dilute forward and reverse primer to 1.6µM concentration
• 2 of 1.5mL vials for forward and reverse master mix 
• 2 unskirted PCR plates (if only running <48 samples, could use same plate) 
• p10 multichannel pipette with a box of tips 
• Repeater pipette, and 0.1mL combitip 
• Ice block(s) 
• Ice cooler 
SECTION: SECTION 3 – BigDye Sequencing Cycle
36. Prep 2 master mixes, one for forward sequencing, and one for reverse sequencing. 
We’ll be doing eighth reactions in a total volume of10 µL (This is a deviation from the manufacturer’s protocol). 

SECTION: SECTION 3 – BigDye Sequencing Cycle
35. Thaw BigDye and keep in ice cooler.  
          a. There are usually several aliquots of 20 µL BigDye in 1.5mL vials. Use those first, unless more   
               is required 
          b. BigDye is susceptible to multiple freeze/thaw. Aliquot out more in 20 µL (or higher, full tray 
              needs 100 µL) when required. *Avoid exposure to light during storage. 

SECTION: SECTION 3 – BigDye Sequencing Cycle
34. Dilute both forward and reverse primers to 1.6µM.  
• Full tray: 16 µL of 10µM primer, with 84 µL of water = 100 µL primer at 1.6µM 
• Small sample size (up to 40 samples): Add 6.4 µL of 10µM primer, with 33.6 µL water = 40 µL primer at 1.6µM 

SECTION: SECTION 3 – BigDye Sequencing Cycle
37. 
 

SECTION: SECTION 3 – BigDye Sequencing Cycle
38. Note: If the purified PCR from ExoSAP-IT does not have sufficient DNA concentration, add more 
purified PCR product and remove water. If going this route, you will need a different master mix ratio. 
SECTION: SECTION 3 – BigDye Sequencing Cycle
39. Mix with pipette or vortex gently, and spin down.  
SECTION: SECTION 3 – BigDye Sequencing Cycle
40. Use repeater pipette to aliquot out 9 µL of forward Master Mix into each well on new unskirted PCR 
plate. 
          a. Repeat step for reverse Master Mix into separate wells. This can be on the same plate as the
          forward sequences if it all fits to a single plate. If not, use a separate unskirted PCR plate. 
SECTION: SECTION 3 – BigDye Sequencing Cycle
41. Add 1 µL of purified PCR product into each well that has the forward Master Mix, and repeat for wells 
with reverse Master Mix.

SECTION: SECTION 3 – BigDye Sequencing Cycle
42. Heat seal plate(s), vortex briefly and spin   down (Do not worry about bubbles). 

SECTION: SECTION 3 – BigDye Sequencing Cycle
43. Run BigDye sequencing thermal cycler program. Runs for about 90 min. 
          a. User name: General 
          b. General → Sanger → BigDye sequencing 
               i. Go to "Edit", to make sure that it has an asterisk beside step 2-4. This means the
                  temperature is ramped up by 1 degree per second 
          c. Program is only on machine 10’s group in our lab
SECTION: SECTION 3 – BigDye Sequencing Cycle
45.  Prep electronic sample sheet for 3500 xL Genetic Analyzer machine while waiting for run to complete 
          a. Go to LAB PROTOCOL BOOK → 13 – Sanger Sequencing 
          b. Open Genemapper Plate Template.xlsx 
          c. Make edits to: 
                     i. Plate Name (cell A4) 
                     ii. Sample Name (cell B6-B102) – leave blank if empty, max 96 samples per 
                         spreadsheet. If more than 1 plate, do each plate separately 
          d. The following stays the same: 
                    i. Assay: Genetics_Lab_Fish_Seq 
                    ii. File name convention: Carp 
                    iii. Results Group: Carp 
          e. Save file(s) as Text(tab delimited) on both project folder, and flash drive. 
SECTION: SECTION 3 – BigDye Sequencing Cycle
44. This can be left overnight, but do not delay too long (no more than a week). If delay is needed, store 
in -20 °C freezer, and protect from light. 

SECTION: SECTION 4 – Post Cycle Sequencing Clean-up
48. CleanSeq <-- Yields better results

Materials needed: 
• Agencourt CleanSeq beads 
• 85% ethanol  
• 50 mL falcon tube(s) 
• ABI plate(s) 
• Multichannel and single pipettes, and corresponding tips (~6 boxes for 48 samples) 
• Repeater pipette
• 5 mL combitip 
• 25 mL reservoir 
• Magnetic plate 
• Lab grade H2O
SECTION: SECTION 4 – Post Cycle Sequencing Clean-up
49. Not recommended to do more than 48 samples at a time, to avoid overdrying of beads mid clean-up
SECTION: SECTION 4 – Post Cycle Sequencing Clean-up
50. Make 85% ethanol in a 50 mL falcon tube. You will need a total of 242 µL of 85% ethanol per sample for this section. Multiply the volume per sample and accounting for pipette error.   
          a. For a full tray --> 21250 µL100% ethanol +3750 µL water = 25 mL 
          b. For 16 samples -->  (16 (samples) + 3 (pipetting error) ) X  242 µL =  4598 µLof 85%   
              ethanol
                            i.4598 µLof 85%   ethanol --> 3908.3 µL pure ETOH and 689.7 µL water

SECTION: SECTION 4 – Post Cycle Sequencing Clean-up
53.  Place plate on magnetic plate, and wait 5 min at room temperature. Magnetic beads will form a 
ring (or 4 lines down the wells, pattern usually alternates on columns depending on the magnetic plate 
you’re using) 
SECTION: SECTION 4 – Post Cycle Sequencing Clean-up
52. Pipette out the required ethanol (42 µL each sample) in a 25 mL reservoir, and use a multichannel pipette to add 42 µL of 85% ethanol to each well 
         a. Pipette up and down 5-10 times to mix solution, discard tips after each use. 
         b. No incubation needed after mixing
SECTION: SECTION 4 – Post Cycle Sequencing Clean-up
51. Add 10 µL of CleanSeq beads into each well with a repeater pipette, aim on the high side of the well 
to prevent contamination.

SECTION: SECTION 4 – Post Cycle Sequencing Clean-up
61. Use a multichannel pipette to dispense 40 µL of water into each well  
          a. Remove any air pockets formed at the bottom of well(s) with a p10 pipette 
          b. Tap plate gently on bench a couple of times, so beads will slide down and go back into the                               
              solution for an easier elution.
SECTION: SECTION 4 – Post Cycle Sequencing Clean-up
55. **Note** Steps 56- 59 can be challenging, but you must be relatively quick, as stalling can cause beads to dry out which will affect the end result. If just starting out, do 2 wells at a time, and slowly ramp up until you are comfortable with 8 wells. 
SECTION: SECTION 4 – Post Cycle Sequencing Clean-up
60. Air dry beads at room temperature for 5 min, avoid overdrying (if cracks are seen, it is overdried. 
You will want the beads to look matte) 
          a. About 1 min into drying: Remove the plate from magnet. Check to ensure there is not an 
              excess of ethanol left in the wells. Pipette out any remaining ethanol. Discard tip after each use. 
          b. Leave plate off of the magnet for the remaining time to dry. 

SECTION: SECTION 4 – Post Cycle Sequencing Clean-up
59. Repeat step 57 and 58 once. 
SECTION: SECTION 4 – Post Cycle Sequencing Clean-up
58. (Plate stays on magnet) Use a multichannel pipette and set to 110 µL to remove all ethanol. 
          a. Lower the tip all the way to the bottom of well and aspirate 
SECTION: SECTION 4 – Post Cycle Sequencing Clean-up
57. (Plate stays on magnet) 
Once all supernatant is removed, use a 5 mL combitip and dispense 100 µL of 85% ethanol to each well. Wait 30 s.

SECTION: SECTION 4 – Post Cycle Sequencing Clean-up
56. Set multichannel pipette to ~65 µL, and take out all the supernatant. Avoid touching or aspirating the 
beads as much as you can.  
          a. It is easier to do 4 at a time or less, until you are comfortable 
          b. Lower the pipette tip to the bottom of the well to aspirate all of the ethanol 
SECTION: SECTION 4 – Post Cycle Sequencing Clean-up
63. Place plate back on magnet for 5 min. 
The unskirted PCR plate should sit on the face of the magnet plate inbetween the holes, not down in the holes. 
     a. This creates bead pellets at the bottom corner of the well instead of a ring, for easier elution. 
SECTION: SECTION 4 – Post Cycle Sequencing Clean-up
62. Incubate plate at room temperature for 5 min, while off magnet. 

SECTION: SECTION 4 – Post Cycle Sequencing Clean-up
64.  Use a multichannel pipette, and transfer 35 µL of the solution into a new ABI plate. Avoid transferring beads! 
           a. If there is more than 1440 min delay to sequencing, transfer to non-skirted PCR plate              
               instead of ABI plate. Heat seal, and store in dark in -20 °C freezer.
               (good up to a month – not tested) 
                     i. If delay is less than1440 min , store in dark in 4 °Cfreezer.  
          b. It is suggested to transfer 2-4 wells at a time by lowering tip to the opposite end of the pellet 
          c. If beads get into your pipette tip, place the solution back into the well and wait for solution to 
              clear up.

SECTION: SECTION 4 – Post Cycle Sequencing Clean-up
65. Seal ABI plate with Nexttec adhesive seal and bring to Wildlife lab for processing in a light blocking container. 
SECTION: SECTION 4 – Post Cycle Sequencing Clean-up
66. Switch out Nexttec seal with the rubber seal and load it on the 3500 xL Genetic Analyzer machine to sequence. 
SECTION: SECTION 5 – 3500 Sequencing Run
75. Enter your plate name, it is recommended to use the same plate name in your .txt file.  
          a. No other changes to settings 
          b. Click “Assign plate contents
SECTION: SECTION 5 – 3500 Sequencing Run
74. Go back to “Dashboard” tab, and select “Create New Plate” 
SECTION: SECTION 5 – 3500 Sequencing Run
73. Under "Consumables Information", make sure there’s sufficient samples left to run your samples. If you are about to run out, reach out to the lab manager to get a new one.  
SECTION: SECTION 5 – 3500 Sequencing Run
72. Go back to the computer, you should be on the “Dashboard” tab on top left.  
SECTION: SECTION 5 – 3500 Sequencing Run
71. Load plate(s) onto 3500.  
          a. Press “Tray” button and wait for machine to bring out the tray platform forward for loading. 
          b. Open the door and load plate(s). 
SECTION: SECTION 5 – 3500 Sequencing Run
70. Open the 3500 data collection software (icon looks like the 3500 machine)  
SECTION: SECTION 5 – 3500 Sequencing Run
69. Once it’s all green, plug in your flash drive (there are more USB slots at back of the tower if the front 
ones are occupied) 
SECTION: SECTION 5 – 3500 Sequencing Run
67. Materials needed: 
• ABI Plate in light protected container
• USB stick
SECTION: SECTION 5 – 3500 Sequencing Run
79. It will take 2 min before the run is initiated, and the estimated runtime is on the top right of the 
screen. 
          a. Each injection takes about 125 min regardless the number of samples within each 
                    injection. 
          b. You’ll want to wait for at least 5 min to make sure the timer continues to countdown 
                    (it refreshes every 5 s). At the 118 min mark, there’ll be movement in the 
                    machine where the first injection is initialized. If the timer continues to countdown after, 
                    the run has successfully begun. You can walk away now. 

SECTION: SECTION 5 – 3500 Sequencing Run
78. Click “Link plate for run”  
          a. If you only have 1 plate → wait 2 min → run window appears → Click on “Start run” 
                    or go to step “b” to add plate B. 
          b. If you have 2 plates, click on “Assign plate contents” on the left column 
                     i. Repeat steps 9-13 
                    ii. It’ll ask to overwrite plate A, or assign to plate B. Assign it to plate B. 
                    iii. Wait a 2 min , then click “Start run” 

SECTION: SECTION 5 – 3500 Sequencing Run
77. If you have your template set up right, you should see samples highlighted with a blue dot, and all 3 
boxes below the tray map are checked with “Genetics_Lab_Fish_Seq” , “CARP”, CARP”.  
          a. If not, do the following: 
          b. In Assay box, if it is empty or not the right assay. Select “Add from library”.  
          c. Under filter, select “Sequencing” → Select “Genetics_Lab_Fish_Seq” 
          d. The other 2 boxes, select “Add from library”, and select “CARP”. 
          e. Highlight all samples, and check the “Genetics_Lab_Fish_Seq” box, along with both “CARP” 
                    boxes. Your samples should now be highlighted in blue. Empty wells should remain 
                    blank/un-highlighted. 
          f. To un-highlight the empty wells, select them and uncheck all 3 boxes 
SECTION: SECTION 5 – 3500 Sequencing Run
76. Click “Import” to import your .txt file 
          a. It might give you some warnings to the import, Click on “Proceed” anyway 
          b. The screen should now show you a map with all your samples to be run 
SECTION: SECTION 5 – 3500 Sequencing Run
80.  Make sure to check back on the machine 60 min later regardless, as the data collection 
software might crash. 
          a. If it does (timer stops counting down, or the software has quit itself), and no error message 
               is given: re-start the plate loading process, and start the run again.  
                    i. Start from the beginning if no data present, or till where it stopped last.  Updated: 2/2/2024 
                              1. To check: Go to Computer → Data → Applied Biosystems → 3500 → Data 
                              → Carp → the last run folder → check each injection (successful AB1 file 
                              should be ~300kB in size) 
                    ii. If injection folders are empty, none of the samples sequenced 
          b. If there is an error message, reach out to Wildlife Lab to troubleshoot if necessary. 
                    i. Note down the error message and timestamp 
SECTION: SECTION 5 – 3500 Sequencing Run
81. Things to note/Troubleshooting steps: 
• Polymer pouch count goes down each time the run is restarted, even though it has not been 
used technically. If you are certain you have sufficient remaining samples to run, proceed to run 
anyway – despite the error message. 

• There are 2 reasons to why the timer stop might stop. If timer stops at about 5 min into the run, chances are the oven is not up to temperature (oven needs to be at 60 °C) 
     o Go to "Dashboard", "oven temp" should be green, and at about 60 °C 
     o Then, check "EPT" tab in the Workflow window, under "Instrument Run Views" and "Flags" 
     o Check to see if lines are still climbing. Takes about ~10 min since you’ve clicked 
         “Start Run”, and timer should countdown at that point.  
     o There’ll be movement at 122 s (switching between buffer A and B, and at about      118 s for first injection)  
     o Overall, this could take up to 15 min from the time you click “Start Run” even if the oven 
         is at temp. So, be patient. 

• If everything seems to be right, and the timer is still not counting down, and you feel you have given 
sufficient time.  
     o It might be a software issue, there is no known reason for the cause, or error messages 
         to tell you it is not running.  last fixed by defragmentation of the computer 6/21/22 
     o Click on “Terminate Run”, abort the entire injection list.  
     o Close the Data collection program, then turn 3500 off. 
     o Shut down the computer. 
     o Then follow instructions to start both the computer and the 3500 back up.  
     o Load the plates on the software again, they should have already been saved. You can 
         load by clicking on “Existing plate” rather than “Create new plate” on Dashboard. 
          ‱ Filter: Sequencing 
          ‱ Type in your plate names, assign plate A (and B) 
          ‱ Start run  

• If new polymer pouch is required: 
     o In the Dashboard homepage, go to Wizards. Select “Refresh polymer” 
     o Go through each step shown on the screen 
          ‱ The lever to load/unload the polymer pouch can be “sticky”, so some force is required  
          ‱ Use a kimwipe with a bit of distilled water to clean the spout. 
          ‱ Check for bubbles, and if all is well, you are set to run the plates 
     o Close the 3500 machine door 

• If you keep getting errors with setting up your assays, and that the oven is not pre-heating. 
The 3500 data collection software may have glitched out. You will have to reboot 
the computer while leaving the 3500 turned on.  
     o After it reboots, everything should go as planned. 

SECTION: SECTION 6 – Collect Results
86. Review quality score on Sequencher. (Ideal is 70% or higher) 
          a. If lower than 60-70%, and scores do not improve after tweaking the sequences, you may 
              need to re-run those samples 
SECTION: SECTION 6 – Collect Results
85. Remove your trays from the machine.  
          a. Remove them from the “jacket” and place the trays in the pile of used trays. 
          b. Wildlife lab will autoclave the rubber tops to reuse, so do not toss them.
SECTION: SECTION 6 – Collect Results
84. Copy the entire run folder to your flash drive, and load to your project folder on S: drive.
SECTION: SECTION 6 – Collect Results
83. The run folder will be listed according to their start date/time, or look for the latest. 
SECTION: Appendix – Successful PCR recipes
87.1. Redband ND1_ND2 region: ND12L and ND12H primers
SECTION: Appendix – Successful PCR recipes
87.2.  Redband dloop region: dloop_Brunelli_F and 28RIBa_DL_R 
SECTION: Appendix – Successful PCR recipes
87.3. 
 

SECTION: Appendix – Successful PCR recipes
88. Thermo Scientific PCR Master Mix (2X) (Cat# K0171) - 50 µL reactions 

SECTION: Appendix – Successful PCR recipes
89. Thermo Scientific Phusion Hot Start II HF DNA Polymerase (Cat# F-549S) - 20 µL reactions

SECTION: Appendix – Successful PCR recipes
88.1. Whitefish CytB region: whitefish2_cytb_765F and whitefish2_cytb_765R 
SECTION: Appendix – Successful PCR recipes
88.2. 
 

SECTION: Appendix – Successful PCR recipes
89.1. Redband mitogenome: EmsermtDNA_F and EmsermtDNA_R (work in progress) 
SECTION: Appendix – Successful PCR recipes
89.2. 
 

SECTION: Appendix – Successful PCR cycling programs
91. Whitefish Cytb
SECTION: Appendix – Successful PCR cycling programs
90.1. Run ND12NEW (General → Sanger → ND12NEW) 
Program located on machines #10-14 
SECTION: Appendix – Successful PCR cycling programs
90.2. 
 

SECTION: Appendix – Successful PCR cycling programs
91.1. Run pstar60 (General → Sanger → pstar60) 
Program located on machines #1-5 
SECTION: Appendix – Successful PCR cycling programs
91.2. 
 

SECTION: Appendix – Successful PCR cycling programs
92. Mitogenome
SECTION: Appendix – Successful PCR cycling programs
92.1.  Run Emser_THERMO (General → Sanger → Emser_THERMO) (work in progress) 
Program located on machines #10-14 
SECTION: Appendix – Successful PCR cycling programs
92.2. 
 

SECTION: SECTION 3 – BigDye Sequencing Cycle
46. 
 

SECTION: PCR Amplification of Target Sequence
11. 
 

SECTION: SECTION 4 – Post Cycle Sequencing Clean-up
54. 
 

SECTION: SECTION 4 – Post Cycle Sequencing Clean-up
48. (no longer in use, Cleanseq beads cleanup is the preferred choice in EFGL)
X-Terminator
Materials needed:
BigDye Xterminator, and SAM solution (both in a yellow Ziploc bag in skinny fridge)
Safety googles
p10 multichannel pipette, and a box of tips
p100 pipette, and box of tips
Wide bore tips
5mL tube
ABI plate(s)
Water
Rubber bands
SECTION: SECTION 4 – Post Cycle Sequencing Clean-up
49.     

 


SECTION: SECTION 4 – Post Cycle Sequencing Clean-up
50. There are 2 separate ways to proceed from here: 
Option A might be easier with fewer samples (less than half a tray), and tends to have better results. Option B would be quicker in general, especially if you have a large number of samples.
SECTION: SECTION 4 – Post Cycle Sequencing Clean-up
51. Option A
Obtain a new ABI plate
Invert Xterminator solution bottle 10x to ensure it is thoroughly mix. Recommended to put on safety googles as Xterminator solution contains Powdex, which can cause serious eye damage.
Use p100 pipette with wide bore tips to add 10µl of Xterminator solution to each well in ABI plate, mix bottle every 2 columns or so.
 Next, use a p100 pipette to add 45 µL of SAM solution to each of those wells. Avoid touching the side of wells to prevent contamination
Use a multichannel p10 pipette and transfer all 10 µLof BigDye sequencing products to ABI plate. Discard tips after each use.
 Do not forget! If you’re doing a partial tray, empty wells/columns need to be filled with 65 µL of water.
  Seal ABI plate with the Nexttec adhesive clear seal. 
 Bring ABI plate and flash drive to Wildlife lab. 
          a.  Remove adhesive seal and seal tray with rubber sealer
          b.  Vortex at high speed for 20 min (you may want to bring your own rubber bands for plate to stay on vortex machine)
          c. Centrifuge for 2 min at 1000 rcf (should be pre-programed)
          b. Load plate to 3500 for sequencing



SECTION: SECTION 4 – Post Cycle Sequencing Clean-up
52. Option B
Obtain a new ABI plate
Invert Xterminator solution bottle 10x to ensure it is thoroughly mix. Recommended to put on safety googles as Xterminator solution contains Powdex, which can cause serious eye damage.
Make Xterminator master mix in 5mL/50mL tube. Multiply volumes by number of samples, accounting for pipette error.
          a.  10 µL of Xterminator solution + 45 µL of SAM solution per sample
Use a p100 pipette with wide bore tip to transfer 55µl of Xterminator master mix
into each well. Make sure to mix the 5 mL/50 mL tube every 1 or 2 columns to
ensure the powdex stay in solution (it settles very quickly). 
Use a multichannel p10 pipette and transfer all 10 µL of BigDye sequencing products to ABI plate. Discard tips after each use.
 Do not forget! If you’re doing a partial tray, empty wells/columns need to be filled with 65µl of water.
 Seal ABI plate with the Nexttec adhesive clear seal. 
 Bring ABI plate and flash drive to Wildlife lab. 
          a. Remove adhesive seal and seal tray with rubber sealer
    d. Load plate to 3500 for sequencing(you may want to bring your own rubber bands for plate to stay on vortex machine)
           c. Centrifuge for 2 min at 1000rcf (should be pre-programed)
           d. Load plate to 3500 for sequencing