University of Pennsylvania
DNA Sequencing Facility

Submission Guide for Sanger Sequencing

Submitting Samples

Step 1: Choose a Sequencing Primer

A template specific primer, or a standard primer may be used. The following standard primers are provided by the Facility at no charge.

Primer Sequence Bases MW Tm
(50 mM Na+) °C
M13/pUC Forward 5′ CCC AGT CAC GAC GTT GTA AAA CG 3′ 23 7018 65
M13/pUC Reverse 5′ AGC GGA TAA CAA TTT CAC ACA GG 3′ 23 7065 61
M13 Forward (-21) 5′ TGT AAA ACG ACG GCC AGT 3′ 18 5533 54
M13 Reverse (-26) 5′ CAG GAA ACA GCT ATG ACC 3′ 18 5502 54
Sp6 promoter/primer 5′ ATT TAG GTG ACA CTA TAG AA 3′ 20 6164 50
T3 promoter/primer 5′ ATT AAC CCT CAC TAA AGG GA 3′ 20 6094 54
T7 promoter/primer 5′ TAA TAC GAC TCA CTA TAG GG 3′ 20 6125 54
T7 terminator 5′ GCT AGT TAT TGC TCA GCG G 3′ 19 5835 57
gt 10 Forward 5′ CCT TTT GAG CAA GTT CAG CCT GG 3′ 23 7031 65
gt 10 Reverse 5′ GGT GGC TTA TGA GTA TTT CTT CC 3′ 23 7052 61
gt 11 Forward 5′ ATT GGT GGC GAC GAC TCC TGG AG 3′ 23 7121 68
gt 11 Reverse 5′ CAG ACC AAC TGG TAA TGG TAG C 3′ 22 6769 62
CMV Forward 5′ CGC AAA TGG GCG GTA GGC GTG 3′ 21 6552 76
BGH Reverse
(pcDNA 3.1)
5′ TAG AAG GCA CAG TCG AGG 3′ 18 5598 56

All primers used at the facility are used at a 1.1μM concentration (except Bacteria 1.5μM). Template specific primers can also be designed and ordered through the Facility for an additional charge.

Step 2: Choosing the Right Concentration

The following concentrations have been found to work the best.

Template Type Size & Template Amount/rxn Template Conc / Vol Primer Amount Primer Conc / Vol
PCR 0.1-1 Kb; 10 ng per 100 bp 1.6 - 16 ng/μl x 6 μl 3.2 pmoles 1.1 μM x 3μl
Plasmid 2-15 Kb; 0.5 μg 80 ng/μl x 6 μl 3.2 pmoles 1.1 μM x 3μl
Phage/
Cosmid/P1
20-100 Kb; 1.0 μg 110 ng/μl x 9 μl 12 pmoles 4 μM x 3μl
BAC 200 Kb; 1 - 2.0 μg 220 ng/μl x 9 μl 12 pmoles 4 μM x 3μl

Step 3: *IMPORTANT* Labelling Your Tubes

All tubes should be marked clearly with the PI and the contact persons name, then numbered in the order placed in the tubes. Here is an example:

labelled tubes

Samples should be submitted in conjoined .2mL strip tubes. No samples will be accepted in 1.5mL tubes. If you happen to forget you will be asked to transfer your samples to .2mL strip tubes at the Facility. Tubes are to be consecutively numbered in such a way as to match the corresponding submission paperwork. The first and second tubes of every strip should be labelled with the PI and user names respectively.

Step 4: Fill Out Online Submission Form

  1. Go to the submission site and log in with your PennKey and password.
  2. Select the grant to be billed associated with your PI (principal investigator). If you do not see any grants coming up, that means that you cannot submit your samples. See your PI/Business Administrator immediately and make sure you are linked to one of your PI's funds. Ask a senior member of your lab for help. See here for more information.
  3. Fill in all of the required contact information.
  4. Select either Low Throughput (≤ 56 samples) or High Throughput by clicking on the button next to these options.
    • Low Throughput (LT) samples: Enter the number of samples. Select the type of samples, and then submit.
    • High Throughput (HT) Samples: Type in your specific plate name. Select your sample type, then submit.
  5. Fill out the form on the next screen. In order for your data entry to be valid it must have a sample name, primer, and template. Sample names must not contain any of the following characters: \ / : * < > | or space. Names may be up to 20 characters long. All Cells besides Special instructions must be filled in. “Auto number” checkbox automatically appends a number to the sample name given. This ensures that all sample names will be unique. “Auto Fill” copies the previous row until it finds a new unique row and then it copies that one. This is useful when filling out a large form. It can be used with auto number to removes any duplicate names for samples. Custom primers can be typed in or be selected as “Included” from the primer pull down menu.
    • Low Throughput (LT) samples: Enter the number of samples. Select the type of samples, and then submit.
    • High Throughput (HT) Samples: Type in your specific plate name, select your sample type, then submit. Alternatively, click “Download CSV Template”, then fill out the form and then import file. Everything if typed correctly will fill all rows needed.
  6. Submit your request.
  7. Print the page that comes up
  8. Log out.

Step 5: Submit your samples

  • At the Facility: Bring the printed page and your samples to the DNA Sequencing Facility in B1 Richards Building. Place your samples in the racks and your sheet in the basket located on the table just inside the door.
  • Use Our Dropbox: Starting September 17th, 2012 you may submit your samples using our dropbox. Just place samples and printed page in a zip-type bag and deposit in the DNA Sequencing Facility drop box located in the lobby of BRB (next to the UPS box). Samples are collected from the drop box twice daily at 11AM and 3PM, M-F, excluding University holidays.

Step 6: Accessing your Data.

Please allow approximately 24 hours for data to be available.

  1. Go to the submission site and log in with your PennKey and password.
  2. Select the “Request Status” tab.
  3. Select the “Details” button next to your samples.
  4. Select “Download Results”. The results will be available if the sequencing has been completed.
  5. Log out.

Evaluating and Editing Sequence Results

The Penn Molecular Profiling Group offers a number of excellent desktop sequencing analysis options for very reasonable licensing fees. Also, see our Software page for links to some free programs for visualizing sequence data.

Sequence calls by the analysis software can include errors. The Facility recommends that all users evaluate and edit the automated sequence calls as necessary by examining the actual color peak data in the chromatograms. The areas one should pay particular attention to are:

  1. Errors in base calls at the start of the sequence due to noise. You may decide to trim away some of the beginning sequence to avoid these error.
  2. Occasional bases missed in the first 80 bases due to compression.
  3. Possible miscalled bases in areas where signal may be low.
  4. Incorrect base calls beyond 600 bases where resolution is deteriorating.
  5. Determining where usable sequence stops, and trimming the remaining base calls.

The results on your chromatogram can be confusing at first glance. There may be more letters than just the AGTC. These other letters are called the IUB codes, and these have very specific meanings. IUB codes are interpreted as follows:

A = Adenosine R = A or G (puRine)
C = Cytidine Y = C or T (pYrimidine)
G = Guanosine K = G or T (Keto)
T = Thymidine M = A or C (amino)
B = C, G, or T S = G or C (Strong-3H bonds)
D = A, G or T W = A or T (Strong-2H bonds)
H = A, C or T N = aNy base
V =A, C or G

Common Causes of Poor or Failed Reads:

  • Too little DNA or primer
  • Too much DNA or primer: leads to stoichiometric imbalance and gradually diminishing peak heights
  • Contamination of DNA
  • Polymerase slippage
  • Using the wrong sequencing primer
  • Sequence read stops abruptly due to secondary structure

The DNA Sequencing Facility is an Abramson Cancer Center Shared Resource that is approved and partially funded by the National Cancer Institute.

abramson-hup

Announcements:
Collection boxes have now been put in the lobby of BRB next to the UPS box and the 1st floor lobby of TRC (near the FEDEX box at the back).  Samples for DNA (Sanger) sequencing can be dropped off into the boxes.  Submission instructions and more information here.


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