Sample Preparation    

The quality of the template DNA directly affects the quality of the DNA sequencing results. In order to generate quality sequencing data, please follow the instructions below to prepare your DNA samples.

		Plasmid DNA purification system in a single tube: - QIAprep Spin Miniprep Kit from Qiagen -  Wizard® Plus SV Minipreps DNA Purification Systems from Promega -  PurElute™ Plasmid Miniprep Kit from Edge BioSystems -  Quantum Prep®Plasmid Miniprep Kit from Bio-Rad
		Plasmid DNA purification system for 96-well plate: - QIAprep 96 Turbo Miniprep Kit from Qiagene - Montage® Plasmid Miniprep96 Kit from Millipore - SeqPrep™ 96 Plasmid Prep Kit from Edge BioSystems
		PCR DNA purification system in a single tube: - AMPure PCR Purification Kit from Agencourt - QIAquick PCR Purification Kit from Qiagen - Zymoclean Gel DNA Recovery Kit from Zymo Research
		PCR DNA purification system for 96-well plate: - QIAquick 96 PCR Purification Kit from Qiagen - ExcelaPure 96-Well UF PCR Purification Kit from Edge BioSystems

 PCR AMPLIFICATION PRODUCTS

It is essential that the DNA is free of contaminants, unused primers, or dNTPs. PCR templates that do not undergo any kind of post PCR clean up are not suitable for sequencing and will yield unuseable sequence data. It is highly recommended that your PCR template is first QC gel checked to confirm that there is a specific product with the correct size.

The EXO/SAP protocol to clean the PCR amplicon frequently damages the PCR primer attachment sites. We recommend that internal primers be used for sequencing because it lowers the risk of poor sequencing results due to the PCR primer attachment being damaged.



 SEQUENCING PRIMERS

ACGT, Inc. provides 50 different free-of-charge universal primers. Specific internal primers can be ordered through ACGT, Inc. To design your own primers, please follow these considerations:

- Perfect matches
- No alternative hybridization sites in template
- No palindromic sequence present, particularly at the 3’ end of primer
- Appropriate length to give Tm of ~55-62°C. Avoid low Tm (i.e. 40-45°C) If Tm is low, make the
  primer longer
- GC "clamp" on the 3' end
- Desirable [GC] = ~50-55%
- Avoid strings of four or more of the same base if possible



 DNA IN GENERAL

1. It is critical that the plasmid template is free of any contaminants including buffers, salts, organics,
    and proteins.

2. Submit DNA in deionized water, not in TE. Buffer components inhibit the sequencing reaction and
    cause failed runs.

3. If possible, use of phenol or chloroform during the purification procedures should be avoided. An
    additional ethanol precipitation is recommended if phenol or chloroform is used.

4. DNA should give an OD260/280 of between 1.7-1.9 and an OD200/260 of about 1.1. Low 260/280
    indicates protein contamination, high OD260/280 indicates possible RNA or residual organics
    contamination. High OD200/260 indicates contamination by organics salts.

5. If your PCR amplification procedure generates a product with a single robust band, column
    purification will suffice. If it generates multiple bands, gel purification of the band with the desired
    size is required.