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Sample Guidelines

Template Types

DNA samples commonly run at the Genomic Core Facility are PCR-generated products, plasmids, bacteriophage inserts or cosmids. Purity is the critical part of sequencing-if the templates are not of the utmost purity the sequence results will be disappointing. For plasmid preps, we find that Sigma, Quiagen and Promega kits provide consistant purity, yielding quality sequence data. For cleaning PCR products, we have had very good results using the High Pure PCR Product Purification kit from Roche, and also with ExoSAP-IT from USB Corpororation.  Gel purification is also a good option. Please be aware ExoSAP-IT does not remove primers from the PCR product, just chews it up. This makes quantifying by spectrophotometry inaccurate. Quantification of  the PCR product will have to be an estimation from running on a gel.

Important!! Do not use Tris/EDTA (TE) buffer to redissolve DNA or primers! EDTA chelates the Mg++ that is required for the sequencing reactions. Phenol from alkaline lysis mini preps is not tolerated at all in the sequencing reaction. Similarly, incomplete removal of cellular components such as RNA, proteins, polysaccharides, and chromosomal DNA will adversely affect your sequencing results. Residual ethanol in the template can also lead to a failed sequencing reaction. A thoroughly dried column prior to the elution of DNA is imperative.

Template Concentration and Volume

The table below shows the concentration needed of template needed.  A picture of the agarose gel the template was run on is also required. Please note the volume of template used in the lanes on the gel.

Template Type

Concentration

Volume

Plasmid DNA, 1 – 10Kb

200 ng/ul

10 ul/reaction

Plasmid DNA, 10 – 20 Kb

400 ng/ul

10 ul/ reaction

PCR Products, 100 – 500 bp

20-30 ng/ul 

10 ul/reaction

PCR Products, 500 – 1000 bp

30 – 50 ng/ul

10 ul/reaction

Large DNA

(lambda, BACs,

 PACs etc.)

200 ng/ul    

1 ug

Bacterial Genomic DNA

500ng/ul

2-3 ug

Sample Information

Write names of templates and primers CLEARLY on top of the 1.5-ml tube exactly as it is on your sequencing order. Data will be posted with primer names, eg. templateA1_primer1.


If any of your samples (primers or templates) are going to be used in multiplereactions, please submit only one tube each containing enough volume for all of the reactions.

Template names are limited to eight characters.  We reserve the right to shorten names exceeding 8 characters.

Do not use punctuation marks (except for a dash), Greek letters or symbols in the template or primer names. The software will not accept them.

If label tape is used, position it so that the tube will still fit into a microfuge. Please don't put Scotch tape over handwritten labels. If the tape comes off, so does anything written underneath it. 

The volume we request is at least double the volume necessary for the concentration required.  This saves time if the sample needs to be re-run.  If the concentration is lower, more volume is required.  If the concentration of your template is significantly lower, quality of the sequence results may also be lower.