Sanger Sequencing

PROTOCOL DUMP PLEASE FORMAT

Designing sequencing primers

Shoot for length of 20-30 nt. UseOligoAnalyzerto look for structure/self dimerization

  • First 100 bps after primer do not give a reliable read (so place primer upstream 100 nt)

  • Sequencing reads are accurate for ~500-1000 bp (you may need multiple primers)

Penn State Core Facility

The Penn State Core facility requires the following for sequencing:

  1. Transfer 2 μL of template to a PCR plate (Sorenson #21970 or equivalently VWR #53550-036). Desired concentrations are:

Plasmid DNA 200 - 300 ng/μL

PCR up to 400 bases 20 ng/μL

PCR 400 – 1000 bases 40 ng/μL

  1. Add 2 μL primer at a concentration of 1 μM. Seal the plate (VWR #89085-360).

(The core facility states 1 μM primer concentration in their directions, but using 100 μM primer concentrations may yield better sequencing results. We need to verify this tip.)

  1. Submit your order on the LIMS system, and follow the directions provided. Drop off the plate to the sequencing facility.

  2. Seal the original plate containing plasmid DNA using foil seal (VWR # ), and store at -20ºC.

TEMPLATES AND PRIMERS MUST BE IN WATER or 10 mM TRIS ph 8.0 (NOT TRIS-EDTA) and each in separate tubes. Do not use 0.2 ml tubes.

Template Requirements per Reaction

5ul @ 200-300 ng/ul for plasmid DNA 5ul @ 20 ng/ul for PCR up to 400 bases; 40 ng/ul for 400 --> 1000 5ul @ 0.4 ug/ul for Large DNA

Primer Requirements per Reaction

5ul @ 1 uM for plasmid & PCR 5ul @ 10 uM for Large DNA

Primers Provided, N/C

T7, T7Term, T3, SP6, M13 Universal, M13-40, M13-47, BGHrevprim, M13 Reverse, M13 Reverse-48, Gal4AD, Gal4BD, Poly dT mix. Inquire about other available primers.

Quintara Bioscience

  1. Login to Quintarabio. User email:salis@psu.edu, password: synbio1980

  2. Under Online Order Quick Select, click on Online Form. And fill out the form with your sequencing samples names, etc. Submit and print form.

  3. Get a PCR strip. Send 500-800 ng of your DNA sample in the tube corresponding to LABEL in the Online Form. Label your tubes with a sharpie. You can premix the DNA and the sequencing primer by adding 5 uL (5 uM) sequencing primer to 10 uL of your DNA sample. If you don’t premix, add 10 uL (5 uM) or 5 uL (10 uM) sequencing primer to an eppendorf tube and 10 uL of each DNA sample separately in a PCR strip.

  4. Get a senior student to show you how to mail samples. Mail before noon.

See http://quintarabio.com/service/dna_sequencing#2 for more info on Quintara DNA sequencing FAQ

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