Column-Pure Blood Genomic DNA Kit

The Column-Pure Blood Genomic DNA Mini Kit is a brief and clean spin column approach for isolation of genomic DNA from as low as a hundred μl of complete blood. The purified DNA is appropriate for molecular biology programs together with PCR, restriction digestion, and lots of extra downstream programs.

Storage of Blood

This package can be used on sparkling or frozen complete blood samples which have been handled with EDTA, ACD or heparin. For quick time period storage (up to ten days), blood ought to be amassed in tubes containing the anticoagulant EDTA and saved at 2-8 °C. For long time storage, blood ought to be amassed in tubes containing popular anticoagulant (ideally EDTA for excessive molecular weight DNA) and saved at -80 °C. To reduce danger of DNA degradation, it’s miles advocated to keep blood for now no longer than three days.

Anticoagulant additives

• zero.five M EDTA pH 8.zero, or ACD
• zero.48% Citric Acid • 1.32% Sodium Citrate •
• 1.47% Glucose For each 1 ml of complete blood sample, upload zero.1 ml of anticoagulant.
• Troubleshooting
• Low yield: Ensure column binding capability of 10 μg isn’t always exceeded.
• • RNA contamination: RNase interest is weak/lost. Add 30 aditional RNase A. Store at 4 °C.
• Sample floats while loading onto agarose gel: Sample includes ethanol from washing.

Protocol

1. A. Before use, observe that Buffer CL can also additionally shape a precipitate upon  storage; if

2. Sample Preparation varies relying at the kind of blood sample:

A. For non-nucleus Erythrocytes (e.g. Human Blood): Harvest ~a hundred μl of blood right into a 2.0 ml centrifuge tube. Add PBS approach to a very last extent of 200 μl. Vortex lightly and allow stand for 1 min at room temperature. If >a hundred μl of blood used, upload 2 volumes of Buffer TBP. Mix very well and allow stand for 1 min till to finish lysis. Spin at 4,000 x g (8,000 rpm) for 1 min. Discard the supernatant carefully. Wash the precipitate with 500 μl TE Buffer times. Spin at 4,000 x g (8,000 rpm) for 1 min all through every wash. The very last precipitate need to seem white. Typical yield is 1-three μg from a a hundred μl blood sample.

B. For nucleus-containing Erythrocytes (e.g. Chicken Blood): Harvest ~10 μl of blood right into a 2.zero ml centrifuge tube. Add PBS approach to a very last extent of 2 hundred μl. Vortex lightly and allow stand for 1 min at room temperature.

C. For solidified blood clot: Weigh zero.1 g of blood. Grind to discover powder below liquid nitrogen. Add 2 hundred μl of PBS answer. three. Add 20 μl of Proteinase K. Mix well. Add 2 hundred μl of Buffer CL. Vortex Gently. Incubate at 56 °C for 10 min. If answer is cloudy, amplify incubation till answer is apparent to finish lysis.

3. If RNA-unfastened genomic DNA is required, upload 20 μl RNase A (10 mg/ml, now no longer provided with package), vortex lightly, and incubate for five min at room temperature earlier than continuing to step five. If very last response extent is >500 μl, growth Proteinase K utilization and/or amplify incubation time.

4. Add 2 hundred μl of a hundred% ethanol to the aggregate and blend very well. Small cloudy insoluble cloth can also additionally seem. This does now no longer have an effect on package performance.

5. Transfer whole tube additives right into a column this is in a 2.0 ml Collection Tube. Let stand at room temperature for 1-2 min. Spin at 8,000 x g (10,000 rpm) for two min. Discard flow-thru withinside the series tube.

6. Add 500 μl of CW1 Solution. Spin at 8,000 x g (10,000 rpm) for 1 min.

7. Add 500 μl of CW2 Solution. Spin at 8,000 x g (10,000 rpm) for 1 min.

8. Discard the flow-thru. Spin at 8,000 x g (10,000 rpm) for extra 1 min.

9. Place column right into a smooth 1.five ml Eppendorf tube. Add 30-50 μl CE Buffer into the middle of the column’s membrane. Incubate at room temperature for two-three min. To growth healing yield, you can strive incubating at 37 °C or 50 °C for two min.

10. Spin at 8,000 x g (10,000 rpm) for 1 min to elute DNA from column. 11. For long time storage, preserve aliquots of purified DNA at -20 °C. 12. Measure DNA amount with the aid of using UV absorption at A260 (1.zero O.D. is equal to 50 μg). Assess DNA best and yield thru zero.7 % agarose gel.

All components (except Proteinase K) can be stored at room temperature. Proteinase K should be kept at 4ºC for short-term or -20ºC for long-term storage. Kit components are stable for12 months at room temperature after received. For maximum stability, store all contents at 4ºC.

Source :

1. NCBI
2. Gentaur