The quality of the DNA template is important to the success of the sequencing reaction.
Single- and Double-Stranded Templates :
We highly recommend the use of the Qiagen series DNA prep kits, as we have found these kits to give consistently high quality sequencing results.
- QIAGEN QIAprep M13 kit: single-stranded templates preparation such as M13
- QIAGEN QIAprep / Plasmid kits: double-stranded templates preparation
- QIAGEN QIAamp: Genomic DNA preparation
- QIAGEN Lambda kits: Lambda phage DNA
- QIAGEN-tip 100: BAC DNA preparation.
It is recommended that the DNA should be resuspended in pure water or Elution buffer (10 mM Tris-Cl, pH 8.5), not TE, as EDTA will inhibit the activity of the Taq enzyme.
PCR Amplicons :
To obtain high quality sequencing data, it is very important that the PCR reaction is specific and strong. If the PCR product is a smear on an agarose gel, or more than one band is present, the likelihood of obtaining a good sequence data is low.
If more than one PCR product is present, use gel purification to isolate the desired product or reoptimize the PCR to obtain a single product.
PCR products must be purified since carry over of PCR primers, dNTPs, enzyme and buffer components from the amplification reaction compete with the sequencing primer and reagents in the sequencing reaction.
Commercially available kits for PCR product purification are listed below:
- QIAGEN QIAquick PCR Purification kit
- ExoSAP-IT reagent (USB) à simple and quick PCR product clean-up
- QIAGEN QIAquick Gel Extraction kit