96-Well Blood gDNA Miniprep Kit

 

 

 

 

 

 

 

 

The EZgene^TM 96-Well Blood DNA Kit allows rapid and reliable isolation of high-quality genomic DNA /viral DNA in a high-through-put 96-well format from a wide variety of samples including fresh, frozen, or anticoagulated whole blood, serum, plasma, bone marrow, body fluids, lymphocytes and cultured cells. Blood DNA is bound to Biomiga’s ezBind matrix while proteins and other unwanted impurities are removed by two rapid wash. Pure DNA is then eluted from the matrix with Elution Buffer or ddH₂O. Purified DNA is suitable for PCR, restriction digestion, and hybridization techniques. The DNA binding capacity per well is 50 µg.

All components of the EZgene^TM 96-Well DNA Kit are stable for at least 12 months from date of purchase when stored at 22-25°C. Buffer BL may form precipitates in cool ambient conditions, Warm up the bottle at 37°C to dissolve before use. Store Protease K at -20°C.

Buffer BL contains chaotropic salts, which may form reactive compounds when combines with bleach, Do not add bleach or acidic solutions directly to the preparation waste, ware gloves and protective eyewear when handling.

 

 

 

 

 

 

 

 

v Laboratory centrifuge capable of at least 5,000 x g equipped with swinging bucket rotor.

v Rotor adapter for deep well microplates

v Water bath equilibrated to 65 °C

v Absolute (96%-100%) ethanol

v Multichannel pipet with tips

v Optional: RNase A stock solution (20 mg/mL)

v Incubator or vacuum oven preset at 65°C

 

 

 

 

 

 

 

Please read the entire manual to become familiar with the EZgene^TM 96-Well Blood DNA kit procedure.

1. Prepare a Protease K stock solution with Elution Buffer and aliquot into adequate portions. Store each aliquot at -20 °C and thaw before use. Each sample will require 25 μL of this solution.

l GD2815-00 Dissolve with 2.5 mL Elution Buffer

l GD2815-01 Dissolve with 10.0 mL Elution Buffer

l GD2815-02 Dissolve with 50.0 mL Elution Buffer

2. Dilute DNA Wash Buffer Concentrate with absolute ethanol as follows and store at room temperature.

l GD2815-00 Add 160 mL (96%-100%) ethanol

l GD2815-01 Add 640 mL (96%-100%) ethanol

l GD2815-02 Add 640 mL (96%-100%) ethanol per bottle

3. Preheat Elution Buffer at 65 °C.

4. Adjust the volume of samples to 250 μL. For samples smaller than 250 μL, add appropriate volume of PBS to 250 μL. For samples larger than 250 μL, split each sample into two 250 μL aliquots and use two wells of the 1.2 mL round well plate for lysis. Load the combined lysates into each well of the 96-Well DNA Plate.

 

Storage of blood samples without previous treatment leads to reduced yields of genomic DNA. For the best result, blood samples should be treated as follows,

l For short-term storage (up to a week), collect blood in tubes containing EDTA as anticoagulant, and store at 4 °C.

l For long-term storage, collect blood in tubes contain an anticoagulant and store at -70°C. Thawed frozen blood sample at 37°C with gently agitation before use.

 

 

1. Dispense 25 μL Protease K (20 mg/mL) into the bottom of each well of the 1.2 mL round well plate. Mark the position of each sample.

2. Add 250 μL whole blood, serum or body fluids to each well of the round-well deep well plate by touching the inside of the well without touching the rims with tip ends (Up to 6 x 10⁶ lymphocytes or cultured cells in PBS can be used in each well).

Note: For sample volumes smaller or larger than 250 μL, adjust the sample volume to 250 μL by PBS (See the Before Starting section on Page 4 for details).

3. Add 250 μL Buffer BL to each sample. Avoid touching the rims of the wells with tips, which might lead to cross-contamination. Optional: Add 5 μL of RNase A per well per 250 μL Buffer BL. Add the Buffer BL/RNase A mix to each well.

4. Seal the round-well plate with caps (supplied) and mix the samples thoroughly by vortexing or vigorously shaking the plate (side to side) for 30 seconds.

Note: Shake the rack side to side. To prevent possible leakage around microtube caps, do not shake the plate up and down.

5. Centrifuge briefly at 2000 x g to collect any samples from caps.

6. Incubate the sample at 65°C for 10 minutes in an incubator or oven. Mix occasionally during incubation by rotating the plate gently.

Note: Do not incubate the sample for more than 20 minutes.

7. Centrifuge briefly at 2000 x g to collect any solution from caps. Remove the microtube caps. Add 260 μL of absolute ethanol (96-100%) to each well.

8. Seal the round-well plate using new caps (supplied).

9. Mix the samples by vortexing or vigorously shaking the plate (side to side) for 1 minute. Centrifuge briefly at 2000 x g to collect any liquid from the caps.

10. Place the DNA plate on top of a 2 mL 96-well Collection Plate (supplied). Mark the 96-Well DNA plate for later identification.

11. Transfer all the samples from Step 10 to each well of the 96-Well DNA plate.

12. Seal the 96-Well DNA plate with a sealing film. Centrifuge at 5,000 x g for 5-10 minutes. Make sure all the samples have passed through the membrane in each well of the DNA plate.

13. Discard the flow-through in the 96-well Collection Plate. Remove the adhesive film cover, and then add 400 μL Buffer KB to each well.

14. Seal the plate with a new sealing film and then centrifuge the plate at 5000 x g for 5 minutes. Discard the flow-through in the 96-well Collection Plate.

15. Remove the sealing film cover, and then add 700 μL DNA wash buffer to each well.

Note: That DNA Wash Buffer is provided as a concentrate and must be diluted with absolute ethanol as indicated on the bottle or page 4. If refrigerated, the diluted wash buffer must be brought to room temperature before use.

16. Seal the plate with a new sealing film and then centrifuge the plate at 3000-5000 x g for 5 minutes. Discard the flow-through in the 96-well collection plate.

17. Remove the adhesive film cover and add 600 μL DNA wash buffer to each well. Place the 96-well DNA plate on top of the 2 mL collection plate, seal the 96-well DNA plate with a new sealing film and centrifuge at maxi speed (5,000 x g) for 10 minutes.

18. Remove the sealing film and incubate the 96-Well DNA plate in a vacuum oven or incubator preset at 70°C for 8 minutes to dry the membrane.

Note: These drying steps are critical for removing the trace amount of ethanol that might otherwise interfere with downstream applications.

19. Place the 96-Well DNA plate on top of new racked microtubes (supplied). Add 200 μL Elution Buffer preheated at 65°C to each well of the 96-Well DNA plate. Incubate at room temperature for two to four minutes or in incubator set at 65°C for one to two minutes.

20. Seal the 96-Well DNA plate with a new sealing film and centrifuge the plate at 5,000 x g for 5 minutes to elute DNA.

Note: The first elution typically yields 60%-70% of the DNA bound to the column. Another 200 μL Elution Buffer can yield another 20%. Volumes lower than 50 μL greatly reduce yields.

 

 

1. Integrated viral DNA or proviral DNA can be isolated by using the same protocol on page 5.

2. To isolate viral DNA/RNA and avoid genomic DNA contamination, cell free samples are recommended. If the viral titer is very low (less than 100 copy per mL), add 10-12 μg of carrier DNA (such as poly dA or Poly dT) to per 250 μL sample. Adjust binding condition by add 280 μL of ethanol instead of 250 μL at Step 7 of the standard protocol.

 

The total DNA yield can be calculated as:

The quality of DNA can be assessed by measuring absorbance at A_260/280. A ratio of (A260/A280) of 1.8-1.9 corresponds to 90%-95% purity.