Q1. How is cDNA generated at ECACC?
ECACC cDNA can be derived from any human cell lines in the ECACC collection. 1-2 x 106 cells are harvested during the log phase of growth and the RNA extracted before reverse transcription is carried out to generate single stranded cDNA.
Q2. What quality tests are carried out?
ECACC checks the extracted RNA for degradation using gel electrophoresis and the presence of genomic DNA is tested for using PCR primers designed to recognise intronic sequences found in genomic DNA.
To check for the presence of cDNA ECACC use PCR using β-actin primers (specific for cDNA) that do not recognise processed pseudo genes (intronless copies of the gene within the genome).
All cell banks that cDNA is generated from are fully authenticated and rigorous in process protocols ensure quality and traceability.
Q3. What concentration and quantity of cDNA will I receive? What format will it be in?
It is not currently possible to quantify total cDNA so ECACC offer a standard single aliquot comprising 20µl of cDNA in a 2ml tube. Alternatively, ECACC provide cDNA samples in 96 well formats as required. Your cDNA will be dispatched on dry ice to your laboratory via courier. Larger quantities of cDNA are available; please contact us to discuss your requirements.
Q4. How long will it take to receive my cDNA?
Normally cDNA will be dispatched within 10 working days of receipt of an order. However, on occasion we may need to extend this time line and in those cases you will be contacted by the ECACC customer support team.
Q5. How stable is the DNA and how should it be stored?
ECACC recommends that cDNA is stored at -80oC when not in use; repeat thawing should be avoided so we recommend making smaller aliquots of the cDNA after the first thaw.
Some laboratories have reported storing cDNA at -20oC and +4oC for extended periods (1-2 months) however there is no stability data for these temperatures currently available. ECACC is currently in the process of carrying out a stability trial, the results of which will be posted on the Culture Collections website as they become available.
We currently recommend using the cDNA within 6 months of manufacture. The date of manufacture can be determined from the lot number. The first two numbers correspond to the year, the second two digits correspond to the month and the last four digits are a consecutive number assigned to the samples we process. So, for example, a sample with lot number 13010001 would have been the first sample processed in January 2013.
Q6. How much cDNA should I use in my assay?
This will depend on your assay; we recommend checking the literature or contacting the manufacturer of the assay you are using before ordering, a standard single cDNA order consists of 20µl of cDNA. If after this you require further help, the ECACC scientific development team will be happy to advise you.
Q7. Why should I purchase my cDNA from ECACC rather than generating my own?
Using ECACC cDNA should save you both time and money. Procuring cDNA from us saves you the time and expense of purchasing cell lines, culturing cells, extracting RNA and generating cDNA only to find the cell line doesn’t contain the gene of interest. ECACC can provide authenticated, quality cDNA in a format to suit you that allows you to progress your research project with confidence.
Q8. Do I get a Certificate with my cDNA? If so, what information will be on it?
ECACC provides certificates of conformity (C of C) on request for cDNA that include the following information
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