Preparing Your Samples

Primer Preparation

DNA Preparation
Reaction Conditions
Troubleshooting Primer and DNA Problems
Sequencing Difficult Templates

Primer Preparation

The quality of your sequencing results is directly related to the quality of your sequencing primer. Therefore we recommend the following suggestions for primer preparation.

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:

Centricon 100 (Amicon)
Qiaquick (Qiagen)
Gel purification

The following preparations 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

Any Questions

For questions regarding DNA Sequencing services please contact Michelle Zanche 276-9997