DNA Sequencing

I’m going to leave this post about sequencing at the FGC up for old time’s sake, but all Glor Lab sequencing should now be conducted following the Agencourt protocol described elsewhere.

A. Sequencing Reactions with BigDye

In each well (total volume=12µl):
1µl             primer (2µM)
0.5µl             BigDye
2.5µl             5X sequencing buffer (in 4°C; green box labelled “Tool box 1”)
6µl             H2O
0.5µl             Betaine
1.5µl             purified DNA

Thermocycler conditions:
96°C            4 min
Cycle 25X:
96°C            10s
50°C            5s
60°C            3 min

B. Pre-sequencing clean-up using Edge plates

  1. Adjust Big Dye reaction product to 100µl by adding 90µl ddH2O to each well
  2. Remove Edge plate from foil packaging (do not throw away!), place on vacuum manifold, and transfer product to Edge plate. Do not let your tips touch the filter membrane to avoid damaging them.
  3. Vacuum dry at 20”Hg until dry – wells should look shiny (~7 mins)
  4. Wash step: add 100 ddH2O to each well on Edge plate and vacuum dry at 20”Hg (~7 mins)
  5. Resuspend DNA by adding 30 µl ddH2O to each well of Edge plate
  6. Put plate back in Edge plate foil packet to keep out of light and let it sit at room temp for 30 min
  7. Pipette product in each well of Edge plate up and down a few times (being careful not to damage the filters) before transferring this product to a new semi-skirted sequencing plate
  8. Cover plate with a foil seal and store in 4C

C. Submitting plate for sequencing at the Functional Genomics Center

  1. Fill out the spreadsheet with a unique sample name for each well by either:

a) filling out the ‘Master’ tab – it’s set up like a normal plate where you can input your sample names and they will be transcribed into the proper upload format in the ‘Upload’ tab
b) directly filling out the ‘Upload’ tab sheet

  1. Save the ‘Upload’ tab sheet as a tab-delineated text file (say ‘ok’ to all the warnings)
  2. Login to: http://fgclims.urmc.rochester.edu/index.html
  3. Under sequencing, click ‘Upload and Import File’
  4. Add the Glor lab account number and upload your file
  5. Print off one copy of your submitted sample list and put it into a zip-loc bag with your samples
  6. Take over to med center Rm G-7814 and place in fridge for pick up. Pick up times are 10:30am and 3pm weekdays.

D. Downloading sequencing results

  1. Login to: http://fgclims.urmc.rochester.edu/index.html
  2. Under sequencing, click ‘Download DNA results’
  3. Find your order/plate and click the ‘Submit’ button
  4. To download all of your sequences, click the ‘Chromat’ button in the top table. Otherwise, you can download your sequences individually from the bottom table by clicking the ‘Chromat’ link in the Download column.

Posted by Julienne Ng – Nov. 19th, 2009

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