Sequencing

DNA sequencing provides a few hundred bases of sequence of a DNA template, which is usually a plasmid or a PCR product. Sequencing is performed by a company (currently Sick Kids TCAG or Bio Basic). The DNA sample, plus a sequencing primer, is delivered to the company and the sequence is returned on their web server a few days later.

A. OVERVIEW/STEPS

1.  Select a suitable sequencing primer (a DNA oligonucleotide). You may have to design and request synthesis of a primer if one is not available. Primer synthesis itself will take a few days.

2.  For each sequencing reaction, add one entry to our lab's Sequencing Record Excel sheet, which is found in the Box Sync > Sequencing folder. Take note of the sequencing record number (format: SQ####) for each of your requests.

3.  Let Emanuel know that you have prepared a sequencing request on the Sequencing Record. He will prepare an order form and email or hand it to you. If you plan to use the Sick Kids Wednesday morning pick-up, do this before Tuesday at 6:00 PM.

4.  Prepare DNA samples and primers as per the company's requirements.

5.  Place the DNA samples, the primer samples, and the order form in a clear zip-lock bag (stored in the cabinets under the water baths). Place the bag in a manila envelope (also stored in those cabinets) and label as indicated below. Bring it to the Sick Kids/TCAG bin outside Room 151 Farquharson before 10:00 on Wednesday morning.

  • Emanuel Rosonina
  • Date
  • Order name (found on order sheet)

6.  Within a few days, Emanuel will let you know that your sequences are ready. They will be placed as text files in the Box Sync folder Recent Sequencing Results in the Sequencing folder. The file name will be the same as the sequencing record number (SQ####.txt). Sequencing trace files (chromatograms) will also be available; ask Emanuel if you'd like to see these.

7.  Analyze your sequence. Usually it helps to prepare a restriction map or to compare with another sequence using nucleotide BLAST. If the sequence produced is on the strand opposite to the strand of interest, produce a reverse complement of the sequencing result for analysis.

 

B. SELECTING SEQUENCING PRIMERS

Each sequencing reaction requires a primer, which is a single-stranded DNA oligonucleotide that matches a sequence upstream of the region of interest. Generally, you will get 650 - 800 bp of quality sequence for each sequencing run, for a primer that is ~100 bp upstream of this region. The sequence obtained will be on the same strand as the sequence of the primer itself.

1.   The diagram below outlines the basics regarding sequencing primer selection. You will get up to 1000 bp of sequence, but note that only 650-800 bp are of high quality. Sequence at the beginning and end of the read is typically low quality, having many "sequencing errors." Therefore, the primer should be situated ~50 bp upstream of the beginning of the region of interest. Note that it is possible to improve the sequence quality of the region close to or distal to the primer by optimizing the sequencing reaction (contact the company).

sequencing01

2.  If you are sequencing part of a plasmid, note that many plasmids include sequences that can be used as sequencing priming sites situated upstream and downstream of multiple cloning sites (polylinkers). Sequencing companies, including Bio Basic, possess common "universal" primers that can be used for sequencing DNA that is cloned in many plasmids. You will not have to send these primers with your sequencing order. Check your plasmid map to see if it has universal sequencing primer regions. Sick Kids and Bio Basic have following universal primers:

Sick Kids/TCAG universal primers

Bio Basic universal primers: M13 Forward, M13 Reverse, pGEX 3’, pGEX 5’, T7promoter, T7 terminator, SP6, T3.

3.  If you are sequencing part of a PCR product, it is okay to use one of the PCR primers as a sequencing primer. However, often primers targeting sequences inside the PCR product give better results.

4.  Our lab oligos stocks may already have a primer that you can use. Check the lab oligo database (Oligos (OLA/B).xls)  in the Box Sync folder.

 

C. DESIGNING SEQUENCING PRIMERS

1.  Generate a restriction map for the region of interest and surrounding sequences.

2.  Design a primer that is 18 - 30 nt in length and has the following properties. You can use an online Oligo Properties Calculator to help you.

  • Melting temperature (Tm): 50ºC - 65ºC
  • No self-complementarity (no regions that will allow it to base-pair to itself or form hairpins)

3.  The primer should be situated at least ~100 bp upstream of the region of interest. Note that the primer sequence will be on the same strand as the sequence that you will obtain. See the following example of a sequencing primer (red) that will provide sequencing results, starting in the shaded green region, and continuing for a total of 650 - 800 nt.

sequencing02-jpg

4.  Once the primer sequence is chosen, fill out the Oligo Order Form. Your primer will be shipped in a few days, and an oligo code name will be provided for the primer (e.g. OLA777). Check the Oligos spreadsheet in the Box Sync > Databases folder.

 

D. PREPARING SAMPLES FOR SEQUENCING WITH Sick Kids / TCAG

Sick Kids/TCAG performs sequencing of plasmids or cleaned PCR products. They do not provide PCR clean-up service like Bio Basic. However, submitting samples to Sick Kids is free because they have a drop-off box in the Farquharson Building. Pick-up is on Wednesdays by 10:00 AM.

  • For Sick Kids/TCAG, DNA samples and primers need to be pre-mixed before submission. Each tube you send represents one sequencing reaction.

Requirements:
1. Use PCR strip tubes to set up your samples. For each sequencing reaction, prepare one tube containing a combination of template and primer as indicated below. Label the tubes with numbers starting with "1".

  • Include 7 µL of template as following:
    • • Plasmids – 200 – 300 ng
      • PCR product (>4 kb) – 100 – 200 ng
      • PCR products (2-4 kb) – 100 – 150 ng
      • PCR products (1-2 kb) – 50 – 100 ng
      • PCR products (<1 kb) – 50 ng
      • PCR products (<500 bp) – 20 ng
      • PCR products (<200 bp) – 10 ng
      • BAC/Cosmid DNA – 600 – 800 ng
  • Include 50 ng of primer in a volume of up to 1 µL (only one primer per tube).
    • If the primer is at 50 µM, add 0.7 µL to the 7 µL tempate
    • If you will be using a standard primer that Sick Kids can provide (e.g., T7, T3, etc.), do not add anything to the template

2. Complete the sequencing order Excel form found in the Box Sync > Sequencing folder. Take note of the sequencing record number (format: SQ####). Let Emanuel know that your samples are ready.

  • In the "Tube label" column, enter the number used to label the tube (1, 2, 3, ...).
  • You can use a 96-well plate if you have a lot of samples. In this case, prepare your samples in the following order, and indicate these labels in the "Tube label" column on the Excel form.
    • Label in this order: A01, A02, A03, A04, A05, A06, A07, A08, B01, B02, B03, B04, etc.

 

E. PREPARING SAMPLES FOR SEQUENCING WITH BIO BASIC

DNA samples for sequencing at Bio Basic can be plasmids (i.e. minipreps), or PCR products that are purified or unpurified. Typically, our lab sends unpurified PCR products for sequencing, and requests that the company perform the purification. If you are sending crude (unpurified) PCR products for sequencing, perform the PCR reaction and check 5 µL of it on a gel before submitting the sequencing reaction.

  • For Bio Basic, DNA templates and primers are sent in separate tubes. Multiple primers can be used with the same template sample.
  • Bio Basic performs PCR product clean-up (Sick Kids/TCAG does not)
  • BIo Basic charges for pick-up, whereas there Sick Kids has a drop-off box in the Farquharson building.

Requirements:
1. For each DNA sample to be sequenced, assign a specific name (even if it will be sequenced with multiple primers). For example, you can name a PCR product with your initials followed by a number (e.g. ER001). This is important for the company to be able to track and identify that DNA sample.

2.  Prepare the DNA templates as follows. Each DNA sample should be placed in 8-strip PCR tubes (or individual microfuge tubes) with the appropriate label (e.g. ER001) clearly written on the tube. Dilute DNA samples in dH2O, not TE.

Plasmid: Prepare 30 - 50 µL of DNA at 100 - 200 ng/µL
Crude PCR product:  At least 45 µL and it must be clearly visible on the gel
Purified PCR product: At least 20 µL at >30 ng/µL

3.  Prepare the primer(s) (unless universal primers will be used) as follows. Each primer can be placed in a different tube within the 8-strip PCR tubes, or in an individual microfuge tube. Make sure it's clearly labeled with the oligo name (e.g. OLA399).

Primers: At least 20 µL of each primer at 10 µM. If starting with a 50 µM oligo stock, take 4 µL of the stock and 16 µL of dH2O to prepare the primer sample. Generally, 1 - 2 µL of the primer prep is  used per sequencing reaction.

4.  If you are using regular microfuge tubes, seal the caps with a small strip of parafilm. If you are using 8-strip PCR tubes, make sure the lids are tightly sealed.

5. Complete the sequencing order Excel form found in the Box Sync > Sequencing folder. Take note of the sequencing record number (format: SQ####). Let Emanuel know that your samples are ready.