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DNA from Buffy coat
I am using the quickgene to extract DNA from buffy coat and I have some interesting numbers after running a sample through the nano-drop.
Nucleic Acid Concentration: 19.2 ng/ul
I blanked the Nanozoom with 2ul of PBS
My preparation of the buffy coat is as follows.
I receive two purple top vacutainers treated with EDTA with between 2-4 ml of whole blood. I centrifuge these tubes at 2500 rpm for 10 minutes at room temperature to separate the layers. I carefully pipette the plasma layer and store it at -80. I then fill two 15 ml conical tubes with cold 10 mM tris 1 mM edta and pipette the buffy coat layer and a few rbc's from one purple top to one of the 15 ml vials. This is allowed to sit for 10 minutes on ice and then centrifuged for 10 minutes at 3000 rpm. The supernatant is poured off and another washing is carried out with 10ml of TE, this is centrifuged again for 10 minutes at 3000 rpm. Repeated washings are carried out until a majority of rbcís are washed away. The pelleted buffy coat is then transferred to a cryo-vial for storage.
If Iím carrying out extraction right away I follow this protocol.
Using one buffy coat from between 2-4 ml of whole blood
240 micro-liters of EDB into a 15 ml conical tube
Suspend buffy coat in 1600 micro liters of PBS
Transfer this to the EDB solution
Add 2000 microliters of LDB
Invert the tube to homogenize
Vortex for 15 seconds
Incubate at 56 degrees C for 2 minutes (this step is skipped due to a water bath on back order)
Add 2000 micro liters of 200 proof ethyl alcohol.
Allow sample to lysate.
An equal amount of this mixture is transferred to each cartridge
DNA whole blood is selected and extraction is carried out.
This is the procedure I am following for my extractions. If you have suggestions or corrections let me know.
|buffy , buffy coat , centrifuge , coat , dna , extraction|
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