How to Submit Sample

Information for Sample Preparation

General Points:

1) Copy Number
Please use a high copy number plasmid. We recommend using one from pUC vectors, pBluescript(R) vectors, pGEM(R) vectors and pTZ vectors, although most other high copy number plasmids will be fine.

2) Host Strain
Most E. coli strains can be used successfully to isolate plasmid DNA, although the strain used can effect the quality of the purified DNA. We recommend using DH1, DH5alpha, and C600 strains. XL1-Blue works well too, but is a slower growing strain. Strains such as the JM series, TG1 and the HB101 series produce large amounts of carbohydrates and should be avoided.

3) Culture Media
Luria-Bertani (LB) broth is recommended as a culture medium. Broths such as TB and 2xYT tend to lead to high cell densities (which may overload the Qiagen puification system we use).

4) DNA Quantitation
We recommend that you assay your template concentration by agarose gel, before submitting samples or proceeding with cycle sequencing. Spectrophotometric quantitation may lead to overestimation of the amount of DNA present.


Specific information for particular Levels of service:


Level 1

Please supply cells from a spun down 5mL overnight culture (see above for recommended conditions). Please supply in a 12ml round-bottomed tubes (from Falcon, Greiner or Sarstedt). Label each tube CLEARLY in a waterproof marker and complete an order form obtained from the PNACL. Please supply relevant primers at 1pmol/ul

 to find out more information on how to provide pellets for this service.

Level 2

The DNA required for automated sequencing needs to be of high purity. We recommend using (preferably) Qiagen, Promega SV+, or Hybaid kits. If you are using a manual method then be sure to remove as much salt as possible from your purified DNA. It is very important to remove all EDTA this is a potent inhibitor of taq polymerase. All DNA submitted must be clearly labelled and have a COMPLETED request form with it. You must also include details of its concentration (this must be close to 0.2ug/uL). Before submission also check it's purity - it must NOT contain protein, carbohydrate or RNA. Also take note of the 'General' points above. Please supply relevant primers at 1pmol/ul.

Level 3

If you are going to supply the completed reaction, please read all the information above. The ABI dye chemistry of your choice can be supplied by the PNACL (please contact us for prices). Pellets must be supplied completely DRY. NO pellets will be accepted if they contain any phenol/alcohol/mineral oil. Please take note of this - there will be long delays in proceesing your samples if PNACL have to clean up your samples prior to loading them onto gels...!


Getting rid of Unincorporated Dyes

Failure to remove unincorporated dyes can severely affect the quality of data obtained from the sequencer as these dyes not only obliterate the first 50-100 bases of the read, but also migrate with longer extension products causing misreading further along the sequence.
 for an example of what can go wrong if excess dyes are present in samples on loading.

There are several methods of removing excess dyes:

1) The most popular method is by ethanol/sodium acetate precipitation:

Remove reaction mix from under oil-layer if necessary and add 2ul of 3M sodium acetate and 50ul of 95% ethanol. Invert the tubes a few times (do not vortex) and leave on wet ice for 10 minutes. Too shorter time will result in the loss of short extension products, too long will result in the precitiation of the excess dyes as well. Spin for 30 minutes at 13000 rpm Remove supernatant and drain tubes upside down on tissue. Wash pellets with 150ul 70% ethanol with gentle agitation. Remove all supernatant and leave to dry.

2) Spin Columns:

This method relies on size-exclusion chromatography, reaction mix is loaded onto preconditioned spin columns and the PCR extension products spun through by centrifugation. There are a variety of commercial suppliers of such columns, for example:

Princeton Separations.
Qiagen.
BioRad.
Millipore.
Edge Separations.

Take care in following the manufacturers instructions particularly paying attention to the recommended centrifugation speeds.

You can make your own spin column using sephadex G50 or similar resin which has been swollen in
 

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