Plasmid DNA

Preparation
It is critical that the Plasmid template is free of any contaminants, please submit DNA in deionized water or in 10 mM Tris. Do not use TE to dilute or resuspended the DNA bacause EDTA inhibits the cycle sequencing reaction. Alpha BioLab recommends using Qiagen miniprep/midiprep or BioRad Quantum miniprep since both methods yield consistent purity of plasmid DNA for sequencing.

Quantitation
We recommend using gel electrophoresis, where the band intensity of a sample DNA is compared to a standard ladder, as the most accurate quantitation. For example, the 1.6 kb band of the popular Invitrogene 1 KB plus ladder contains 8% of total amounts of DNA presents in the ladder mixture, and can be used as quick reference for estimating the concentration of your template.

Please provide DNA in the concentration range of 100-500 ng/µl and in the amount of at least 1.5-2 µg. Extra amount of DNA ensures that we have enough sample to do a re-run in case the first reaction fail. Samples with concentrations that are not in this range or failing to provide us with enough template to do the reaction will cause delays.

PCR fragments

Preparation

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 useable sequence data. It is highly recommended that your PCR template is first observed on a gel to confirm that there is a specific product with the correct size. The Qiagen Gel extraction kit or PCR cleanup kit can be used to remove all of the unwanted products from your template.

Quantitation

Gel electrophoresis is the method of choice for PCR fragment quantification. For each reaction, please provide 10 ng/100 bases, and at least 20 ng/µl solution in deionized water. Please provide at least 10 µl to allow for any possible re-run.

PRIMERS

Preparation

Primers supplied by customers should be desalted or purified. Crude primers generally do not work well for sequencing. Here is a list of common primers that are available for sequencing at no charge.

M13 forward GTA AAA CGA CGG CCA GT
M13 reverse GGA AAC AGC TAT GAC CAT G
T3 ATT AAC CCT CAC TAA AGG GA
T7 AAT ACG ACT CAC TAT AG
BGH reverse TAGAAGGCACAGTCGAGG
SP6 GAT TTA GGT GAC ACT ATA G

Please supply primers at concentration of 10 µM (~10pmole/µl =60 ng/µl) in deionized water at volume of greater than 20 µl. If you are sequencing a PCR product, make sure that the excess primers from PCR reaction is removed. We recommend that you use primers that are internally nested in the construct.