Sample requirements

Sequencing Chemistry

Sequencing reactions are performed using Applied Biosystems 'BigDye' Terminator version 3.1 chemistry (BDTv3.1). This version provides a significant improvement over earlier versions in that it generates better sequence data closer to the primer as well as longer readlengths.

Sample Contents

We require samples in the form of single-reaction 'cocktails' containing template DNA plus primer made up with sterile Milli-Q water to a total volume of 12µL.

Sample Tubes

If you have 8 or more samples, please supply all of them in 8-strip 0.2mL PCR tubes with 8-strip dome caps.

If you have less than 8 samples, please provide them in any combination of the following: -

8-strip 0.2mL PCR tubes with 8-strip dome caps
1.5mL microcentrifuge tubes
0.5mL microcentrifuge tubes
0.2mL PCR tubes

Tube Labelling

For 8-strip PCR tubes

Please label the first and last tubes in each strip with their respective sample numbers (e.g. 1 & 8, 9 & 16 etc). Also include your initials somewhere on the strip.

For microcentrifuge tubes

Label the lid of each tube with its respective sample number (e.g. 1, 2, 3 etc.) and include your initials on the side of each tube.

Sample Delivery Form

Please fill in the sample delivery form to accompany your batch of samples.

Filenames: Please use letters, numbers, and the following punctuation only -_(){}#.+ with no spaces. A maximum of 20 characters may be used.

DNA Amount

The suggested amounts of template DNA to use per reaction are shown below (from BigDye v3.1 protocol). These amounts provide a good starting point - further optimisation may be required in some cases.

Template DNA	Size (bp)	Amount per reaction
PCR Product	100 - 200	1 - 3 ng
	200 - 500	3 - 10 ng
	500 - 1000	5 - 20 ng
	1000 - 2000	10 - 40 ng
	> 2000	20 - 50 ng
Single-stranded		25 - 50 ng
Double-stranded		150 - 300 ng
Cosmid, BAC		500 - 1000 ng

Primer amount - use 3.2 pmol per reaction

For GC-rich templates (or where secondary structure might be an issue) please include 1uL DMSO (dimethyl sulphoxide) in the 12µL sample volume.

Results are highly dependent on the quantity and quality of DNA

Quantity

It is strongly recommended that you quantitate at least a subset of your DNA samples by spectrophotometer (the preferred method).

Alternatively, you may wish to quantitate your DNA by agarose gel/ethidium bromide staining - i.e. by comparing against known amounts of DNA (e.g. size markers).

Another possibility would be to provide your own series of template dilutions for sequencing and use the results obtained to establish a 'rule-of-thumb'.

Quality

The capillary instrument we use is more sensitive than the older slab-gel systems!

Many of the commercial spin or vacuum purification kits produce DNA that is suitable for DNA sequencing. Unless carried out carefully, some other methods such as alkaline lysis/phenol/chloroform extraction or loose silica gel ('glassmilk') will likely produce poorer results. Please contact us for recommendations.

Similarly, primers should be purified to 'PCR/sequencing grade' to achieve best results (check with manufacturer).