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DNA Extraction

 

What method do you use to extract the DNA?

We currently out-source DNA extraction to our experienced ISO 9001 certified European partner, their extraction chemistry is based on magnetic bead technology. The magnetic separation eliminates centrifugation and phenol extraction steps and allows us to fully automate the whole process. There are three basic steps: the first step involves the lysis of cells and binding of DNA to the magnetic particles. The DNA is then washed three times and eluted.

 

What is the typical DNA yield from a 10ml blood extraction?

The yield is dependent on the amount of DNA containing cells in the sample but is typically 300-500ug of DNA from a 10ml blood extraction, 15-25ug from a 2ml blood extraction, 4-6ug from a 200ul blood extraction.

 

Are you able to extract DNA from small volume of blood?

Yes, we can extract DNA from as little as 10ul of blood. A minimum of 20 samples is required for small volume (less than 2ml blood) extraction.

 

Are you able to extract DNA from buccal swabs/FTA paper?

Yes, our extraction method is able to extract high quality DNA from buccal swabs and FTA paper provided the swabs and paper have been stored appropriately according to the manufacturer's instructions.

 

Are you able to extract DNA from cell lines?

Yes, our extraction method is able to extract high quality DNA from frozen cell pellet.

 

What is the quality and stability of the extracted DNA?

The extraction methods produce purified PCR-ready genomic DNA. The typical ratio of absorbance at 260nm and 280nm of the extracted DNA is between 1.7 and 1.9 and the expected size the extracted DNA is more than 20Kb. Typical results from using the extraction method are shown in the following figures.

Table 1:

Sample ID:	Concentration DNA (ug/ul)	Total yield (μg)	Ratio A260/A280	Sample material
1	0.045	9.0	1.77	1ml Whole blood
2	0.067	13.4	1.78	1ml Whole blood
3	0.011	2.2	1.89	200μl Buffy Coat
4	0.050	10.0	1,79	200μl Buffy Coat
5	0.048	9,6	1,78	1ml Whole blood
6	0.047	9.4	1.78	1ml Whole blood
7	0.069	13.8	1.81	1ml Whole blood
8	0.045	9.0	1.83	1ml Whole blood
9	0.032	6.4	1.80	1ml Whole blood
10	0.039	7.8	1.82	1ml Whole blood
11	0.054	10.8	1.75	1ml Whole blood
12	0.077	15.4	1.82	1ml Whole blood
13	0.041	8.2	1.83	1ml Whole blood
14	0.041	8.2	1.81	1ml Whole blood
15	0.043	8.6	1.84	1ml Whole blood
16	0.038	7.6	1.74	1ml Whole blood
17	0.062	12.4	1.81	1ml Whole blood
18	0.026	5.2	1.83	1ml Whole blood
19	0,034	6.8	1.87	1ml Whole blood
20	0.054	10.8	1.89	1ml Whole blood
21	0.028	5.6	1.78	1ml Whole blood
22	0.109	21.8	1.53	1ml Whole blood
23	0.032	6.4	1.88	1ml Whole blood
24	0.083	16.6	1.82	1ml Whole blood
25	0.036	7.2	1.80	200μl Buffy Coat
26	0.022	4.4	1.89	200μl Buffy Coat
27	0.114	45.6	1.84	200μl Cultured cells
28	0.162	32.4	1.83	200μl Cultured cells
29	0.087	17.4	1.82	200μl Cultured cells
30	0.245	49.0	1.65	200μl Cultured cells
31	0.041	8.2	1.83	200μl Cultured cells
32	0.074	14.2	1.82	200μl Cultured cells

Yields of DNA extracted from different starting materials

 

Figure 1: The reproducibility of our extraction chemistry on automated liquid handling systems. Each sample was extracted in triplicate to show that the yield of each sample is similar and high molecular weight DNA is obtained.

 

 

Figure 2: A cross contamination test. Blood samples and water were arranged alternatively in automated liquid handling system for extraction. After extraction, PCR reactions amplifying a fragment of the CFTR gene (exon 4) were set up using the eluate of all the samples. PCR product was only detected from the eluates from the blood samples and not the water.

 

 

What is the stability of the extracted DNA?

The genomic DNA is stable long term when buffered in Tris, pH 8 and stored at -20 degrees C.

 

How should I send the samples to Source BioScience LifeSciences?

Recommended shipping conditions for various sample types are as follows:

• Cells, dry ice
• Blood, dry ice
• Buccal swabs, dried at room temperature
• FTA paper, dried at room temperature.
• It is essential that in addition to a signed agreement and purchase order number that we also get the following information from you for each sample you send:
• List of samples delivered
• Sample source (human, animal etc)
• Sample type (EDTA; citrate; heparin; buffy coat; buccal swab etc)
• Sample volume
• NB: Material which is known (or suspected) to be infectious must be labelled as such and Source BioScience LifeSciences must be told about them in advance of receipt.

What is a typical turnaround time for the DNA extraction?

We routinely complete 200 DNA extractions in a week although higher throughput extraction can be organised on request.

 

How will I receive the extracted DNA?

The extracted DNA is typically buffered in 10mM Tris, pH8 although this can be flexible according to your needs. The volume of buffer used is as follows:

• 10ml blood sample, DNA returned in 1ml buffer
• 200ul - 2ml blood sample, DNA returned in 200ul buffer
• 10-50ul blood sample, DNA returned in 50ul buffer
• FTA Paper/buccal swab sample, DNA returned in 50ul buffer

 

We can supply the DNA in a number of formats. However, Source BioScience LifeSciences recommends the use of 2-D bar coded tubes. This allows greater sample security. Before accepting our quotation for DNA extraction, you will need to indicate the preferred format to receive the extracted DNA.

 

What happens if I want Source BioScience LifeSciences to do some genotyping following extraction?

We strongly recommend the DNA is stored in 2-D bar coded tubes. The samples will then be diluted appropriately for genotyping and will be passed to the relevent team. You will need to ask us for a follow up proposal of work or we can bundle the genotyping into a single project price with your extraction.

 

Can you help me with secure storage and retrieval for my extracted DNA samples?

Yes. Source BioScience LifeSciences has a DNA banking storage and retrieval system that includes making a mirrored off-site store of your samples. Please contact us for more details.