The Huck Institutes of the Life Sciences

Sanger sequencing sample preparation recommendations

How to prepare your sequencing samples for traditional Sanger sequencing or high throughput sequencing

Please submit all materials at the correct concentrations.

With up to 100 samples a day, we do not have time to do dilutions.

 

Traditional Sanger sequencing

Please read all information at the dnaLIMS site regarding submission of samples.

Our additional recommendations include the following:

 

DNA templates

Please submit 5μls for any template, and at the following concentrations:

  • 20ng/μl for PCR products up to 400 bases
  • 40ng/μl for PCR products 400-1000 bases
  • 100 to 200ng/μl for PCR products between 1000 and 3000 bases
  • 200 to 400 ng/μl for plasmids up to 10K
  • 400ng/μl or greater for plasmids 10K-100K
  • Inquire about templates greater than 100K

 

Primers

  • Submit 5μl of primer at a concentration of 1μM for all PCR products and plasmids less than 10K
  • For those greater than 10K, submit 10μl of primer at a concentration of 10μM

Please check primer sequences for the primers we supply to make sure your promoter sequence is the same.

 

Submitting plates for sequencing

  1. To submit plates for sequencing, please purchase plates and seals from one of the following companies:

    Denville (these are the same as the old PGC plates and seals):

    • PCR plates #C18096-10
    • PCR plates #C18096
    • Seal #B1212-5
    • Foil Seal (may work better for those mailing plates to us) #B-1212-6

    VWR:

    • PCR Plates #53550-036
    • Seal #89085-360
  2. Please add 2uls of template at the following concentrations to the plate (note: this is only for plates, not for regular submissions):
    • Plasmid DNA: 200-300ng/μl
    • PCR up to 400 bases: 20ng/μl
    • PCR 400-1000 bases: 40ng/μl
  3. Add 2uls of primer from a solution of 1uM to the plate. (Unless you are using our primers. please inquire about this.)
  4. Submit your samples on the LIMS System using the Upload and Import File Feature.
    • Follow the instructions to download the file.
    • Type in your sample names.
    • Upload the file and print out a copy of the order to submit with your plate.

 

Some things to remember when submitting plates:

  • If you need primer added to your plate, please note this in the comment section of your order form. If you do not list this, we will have to e-mail you and this will delay the plate being set up.
  • If you need multiple primers added to your plate, please place samples with the same primer in one section of the plate. For example, if you have samples that need T7 and SP6, place all samples that need T7 in one area of the plate and all samples that need SP6 in another area of the plate. We will not add primer if they are scattered all over the plate.
  • Because we receive many plates every day, you must label your plates. Please put your name and/or lab name AND the Order Number on your plate. Please label the plate, NOT just the seal. We will NOT run your plate if you have written the order number ONLY on the seal.
  • Make sure you print out an order form and submit it with each plate.
  • When you load your plate start at A1 and go down to B1, C1, D1 etc and then go up to A2. Do not go across the plate because this will cause the sample names to be imported incorrectly. Also if you have skipped wells (wells with no sample in them) please make sure you put something in for the sample name (such as blank or empty). If not this will cause all the sample names to be moved up and imported incorrectly.
  • Drop off your plate in accordance with the sample drop-off guidelines in Room 413 Chandlee between 8 am and 4:30 pm.
  • Plates are not guaranteed to be run the same day that they are brought to us.