McClean: Sequencing Colony PCR Product

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Contents

Overview

This protocol is for sequencing the product of a yeast colony PCR.

Materials

  • Colony PCR product
  • DNA Clean & Concentrator Kit (Zymo Research)
  • Sequencing primers

Protocol

  1. Check your colony PCR product on a gel.
  2. If you have one clear band, follow the Zymo Research DNA Clean & Concentrator Kit instructions for cleaning up your colony PCR reaction.
    1. This is a PCR reaction, so add 5 volumes of DNA Binding Buffer in Step #1.
    2. Elute your DNA (Step #5) in 10μL of H2O (NOT Elution Buffer).
  3. Use 1μL of this DNA product to check concentration on the nanodrop.
  4. Prepare the solution to be Sanger Sequenced at the UW-Biotech Sanger Sequencing Facility. These instructions are for submitting a "Discount- Strip Tube order". The Sanger Submission, the Guide to successful sequencing, and the Materials and methods pages may be helpful. WARNING- These instructions were made specifically for sequencing PCR product, and were written by Cameron Stewart who (at the time of writing this) is a complete novice. At least skim through the linked pages. The following instructions were given to me when I asked the Sanger Sequencing staff what to do because I found the online instructions confusing.
    1. Fill a .2ml PCR Strip Tube (not a 1.5ml Eppendorf tube) with 10ng/100bp of your pcr product (or 0.2μg/6500bp of plasmid according to Guide to successful sequencing). For example, if your PCR product is 580bp, then you want 58ng. So, if your DNA concentration was 25ng/μMol, then you'd want to insert 58ng x 1μL/25ng = 2.32μL of your PCR product into the tube.
    2. Fill the tube with 10 picoMoles of one primer. For example, if your primer is at 10μM concentration, then insert 10 x 10-12 Moles * (1L/10*10-6moles)=10-6L=1μL.
      1. The Guide to successful sequencing says "It is important to limit primer amount to 5 pmol when sequencing PCR fragments," but in person I was told to use 10pMoles. When I asked about this contradiction, they said 10 pMoles is good, but 5 pMoles is also good... >_<
    3. Bring the total volume to 20μL with DI water. For example, if your DNA + primer volume is 3.3μL, then insert 16.7μL of water.
      1. Some of the webpages say the total volume should be 24μL. In person I was told 20μL. When I asked about the contradiction, they said 20μL is good but 24μL is also good... >_<
    4. Label the strip tubes individually. Then wrap them in foil and on a peice of tape on the foil write your PI's name, your name, the date, and the name of the set of samples. (The foil itself isn't special, a bag could be used too, anything to keep them together and labelled.)
    5. Go to the Biotech facility (room 2360 at the time of writing this) and log in to the computer. If the tubes are individual and not connected select Discount Sequencing for $10/sample. Discount Sequencing-Striptube, $7.50/sample, is for connected 0.2ul PCR tubes. Place the printed order on the marked plastic shelf and leave your labelled sample in the fridge.
    6. If you are still confused, the people at the facility are extremely patient and helpful. Talking to them in person may be best.

Analyze Results

  • Cameron J. Stewart 11 April 2016 I've found these to be helpful
    • BioEdit [1] is a free tool to view the sanger sequencing results
    • The NIH's "Align Sequences Nucleotide BLAST" [2] is a useful tool to see how the sanger sequence aligns with a map. For example, if your Sanger Sequence is of a PCR amplified section of a genome, enter your Sanger Sequence as the Query and a map of the genome as the subject.

Notes

Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input! Please sign your name to your note by adding '''*~~~~''': to the beginning of your tip.

  • Megan N McClean 15:25, 25 March 2013 (EDT) Any method of cleaning up the DNA should be fine. In my hands, the Zymo Research kit seems to work the best. Make sure to elute in WATER and NOT elution buffer in the final step. This seems to help a lot.
  • The old protocol, relevant to the procedures at Princeton, said (after using 2μL to measure the concentration at the nanodrop): To the 8μL of remaining DNA, add 4μL of primer at 8pmol. (Our lab oligos are at 10μM, so to get the desired 8pmol of oligo, dilute our oligos 1:5 in sterile water for a final of 2pmol/ul and add 4μL to the 8μL of DNA to be sequenced).

References

Contact

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