Preparing Your Samples

Primer Preparation

The quality of your sequencing results is directly related to the quality of your sequencing primer. Therefore, we recommend the following guidelines for preparing sequencing primer.

Guidelines for Preparing Sequencing Primer

  • Primer Length—Minimum 18bp (20-22bp recommended)
  • Primer Tm—Should be around 55 degrees
  • GC content—50% (more critical on the 3' end)
  • G/C "clamp" on 3' end
  • Avoid hairpin loops within primer
  • Avoid long stretches of a single base (3 or 4)

Preparing DNA

The quality of your sequencing results is directly related to the quality of your template. Therefore, we recommend the following suggestions for sample preparation:

Host Strain Recommendations

Host strain selection is important and if possible it is best to use a highly recommended host strain. Please refer to the chart below before choosing a host strain.

Highly Recommended Recommended Not Recommended
DH5 alpha JM109 JM101
HB101 XL-1 Blue MV1190

Methods for DNA Template Preparation

Template should be protein, RNA, and contaminant free. We recommend the following preps:

For Plasmid DNA

  • Qiagen Mini Prep Kits
  • ABI Prism Mini Prep Kit
  • Alkaline Lysis/PEG Precipitation
  • Promega Wizard (although modifications required, ask for details)
  • Promega Wizard SV

For PCR Products

Preparations that are NOT Recommended

  • Bio 101 GeneKleen
  • Cesium Chloride (unless dialyzed extremely well)

Allowable Contaminant Concentrations in DNA Templates

Values greater than those listed in the chart below may significantly reduce the quality of your sequencing results.

Contaminant Amount Tolerated in Sequencing Reaction
RNA 1ug
PEG 0.3%
NaOAc 0.5mM
Ethanol 1.25%
Phenol/Chloroform 0%
CsCl 5mM
EDTA 0.25mM

Questions?

For questions regarding DNA Sequencing services, please contact Michelle Zanche, (585) 276-9997.