In an experiment, I prepared serial dilutions from fresh human blood samples (EDTA tubes) using TE buffer. Now I need to know if the white blood cells were intact or not in the diluted blood (there are no samples available today). The TE buffer used was made by mixing 10mL Tris-HCl (1M pH 8.0) and 0.2mL EDTA (0.5M) in 990mL Milli-Q water. Any suggestions or references would be appreciated.
White blood cells remain intact in TE buffer.
In DNA extraction from blood first step is removing red blood cells, while keeping white cells. In many protocols TE is used for that purpose.
White blood cells are easily aggregated in hypotonic buffer during isolation from whole blood. I am not sure your goal, but if you need intact cells during the serial dilutions, you should use an isotonic buffer (I would add NaCL at the final 150 mM in TE). Quality of RNA and DNA from aggregated /dead cells are lower than that from intact cells.
Thanks for valuable input! This will be useful in the future.
The goal was to prepare dilutions for a sensitivity/"drop-out" study for a forensic STR DNA typing kit. Target range was 1000-25pg of DNA in the final PCR reaction. Drop-out of one allele in a heterozygous loci is caused by stochastic effects in the sampling of molecules from low concentration samples (i.e. some chromosomes/loci might not be present in the aliquot taken from the extract to the PCR reaction due to chance). A drop-out might lead to erroneously designating a heterozygous loci as homozygous. These tests are used to estimate a peak height threshold where the risk is small. However, there is a difference to use "intact cells" with the chromosome pairing still intact, as compared to "naked DNA" where the pairing is broken. Naked DNA in high concentration diluted (in several steps) to very low concentrations can lead to increased stochastic variation, therefore it is recommended to dilute the body fluid (blood) before extracting the DNA. This procedure is unfortunately much more laborious then simply using naked DNA (it is e.g. difficult to estimate the concentration after extraction).
I am still interested in a reference confirming the effect TE has on white blood cells.
TE will only lyse red blood cells and not the white blood cells...u can also see
http://www.iarc.fr/en/publications/pdfs-online/wrk/wrk2/standardsBRC-8.pdf
Hope it helps!
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Biology
- Molecular Biological Techniques