Small and large scale preparations
The first step in any study investigating genetic differences in an organism of interest is the purification of genomic DNA from blood or other tissue type. We currently offer purification from saliva, blood and rodent tails. Should you be interested in preparing DNA from another tissue type, then please don't hesitate to contact us.
Technology
We use our own in-house silica based systems for blood, buffy, saliva, buccal swab and tissue extractions. We have optimised this system to produce high quality, high yield DNA which has been shown to be suitable for long term storage and more importantly the process does not require a precipitation step. The removal of the requirement for DNA precipitation means that it is impossible to lose pellets, resulting in extremely high success rates.
We conduct all extractions manually. The rationale for this is that robotic handling of high viscosity genomic DNA is notoriously difficult. It also allows us to deal with old and clotted blood effectively. This also ensures that both small and large scale preparations are prepared the same, and thus are of the same quality. However, if you have a preferred technique then we will of course be happy to use this.
How do we do it?
A brief pictorial flow of the process with an explanation of the stages is shown below.
1. Blood Transfer to the Lysis tube
The whole blood is transferred into a solution containing Guanadinium Isothiocyanate and Proteinase K. The combination of the chaotropic salt and the protease breaks down the cells to release the nuclear material and inactivate cellular nucleases.
2. Transfer to the silica column
The blood lysate is then adjusted for optimal column binding conditions by the addition of Ethanol. A number of washes follow this step to remove all unwanted comtaminants.
3. Elution of the genomic DNA
The DNA is then eluted to a fresh tube in 10mM Tris pH8.0 and transferred to a 2D datamatrix barcoded tube
4. DNA quantitation and normalisation
DNA is quantitated and normalised to the customers desired concentration.
5. Final QC check by PCR
Unlike other providers KBiosciences recognises that a functional test of the DNA quality adds a level of confidence in the purified material. We perform a dilution series test using a couple of in-house KASPar assay to ensure the DNA is of the highest qulaity before sending out to the customer.
An example of data from this test is shown below.
Tracking
We employ a barcode tracking system that ensures your complete confidence
in our service. Upon receipt of either plates or individual tubes a barcode
is applied (if one doesn't already exist). This is immediately entered into
our LIMS. Further to this, any subsequent tube transfers are also treated in
the same manner. The barcode tracking information is then supplied back to the
customer along with the purified DNA.
Quality Control
KBiosciences strives for the highest possible quality in its purification
service. Each sample is subjected to the following rigorous QC criteria
- Each extraction is run on an agarose gel to ensure samples are high molecular weight
- OD260/280 must be greater than 1.7.
- Each purified DNA is supplied with a concentration determined by PicoGreen analysis
- For sample numbers >48 the DNA is included in a KASPar genotyping reaction. It must perform well giving a clear genotype to meet QC criteria.