RBC Bioscience MagCore HF48 User Manual page 45

Running Time:
42min (without DNase I treatment ; starting volume: 200 µl)
68min (with DNase I treatment ; starting volume: 200 µl)
Preparation before using
1. β-Mercaptoethanol (β-ME; not provided) must be added to RB Buffer before use. Add 10μl of β-ME per 1 ml of RB Buffer.
2. Recommended Step: DNA residue degradation. Prepare DNase I (RNase-free) working solution according to the table below.
Add 10μl DNase I with 190μl DNase reaction buffer (1X) in the 1.5 ml screw tube (not provided) and place it into well 3 of T-Rack.
Healthy Whole blood
Up to 400 μl
3. RNase-free DNase I is not including in MagCore total RNA Whole Blood Kit, we recommend to use RBC RNase-free DNase I
(Cat#DN036 or Cat#DN096) for genomic DNA treatment. For product information, please contact RBC Bioscience distributor.
We also recommend to use RNase-free DNase I enzyme(1U/μl) of Novagen (Cat#69182-3). Please contact local Merck branch
office or distributor for product information. 1X DNase Buffer can be prepared as following:
1X DNase I reaction buffer
10 mM Tris, pH7.6 ; 2.5 mM MgCl
Fresh Whole Blood Protocol
Without DNase I treatment
1. Add 1 volume of human whole blood with 3 volumes of RBC lysis Buffer in an appropriately sized tube (not provided) and mix
by inversion. Do not vortex.( For example, add 1200μl of RBC lysis Buffer to 400μl of whole blood.)
2. Incubate the tube for 10 minutes on ice and invert 2~3 times during incubation.
3. Centrifuge for 3 minutes at 500 x g (2,500rpm) at 4°C and completely discard the supernatant.
4. Add 500μl RBC lysis Buffer to the cell pellet. Resuspend cells by vortex briefly.
5. Transfer the suspended cells to the MagCore Sample Tube.
6. Centrifuge for 3 minutes at 500 x g (2,500rpm) at 4 °C and completely discard the supernatant.
7. Add 200μl RB buffer (contain β-ME) to the white pellet and mix by vortexing. (can storage up to 1 month at -80°C)
8. Place the prepared Sample Tube into well 4 of T-rack.
9. Put the Elution Tube into well 1 of T-Rack and the Tip Plus Holder Set into well 2 of T-Rack.
10. Run Code.601 program at MagCore® and select Remove Genomic DNA (2)NO.
With DNase I treatment
1. Follow step 1~9 of without DNase I treatnent protocol to prepare
whole blood cell sample.
2. Be sure to place the 200μl DNase I mixture (in 1.5 ml screw tube)
into the well 3 of T-Rack.
3. Run Code.601 program at MagCore® and select Remove Genomic
DNA (1)YES.
DNase I
10 μl
; 0.1 mM CaCl ; in DEPC water, autoclave.
2 2
DNase buffer 1X
190 μl
Well
Elution Tube
Tip / Tip Holder
DNase
Sample Tube
MagCore®
42