Bacterial Genome DNA Extraction

Summary

Bacterial genomic DNA extraction can be applied to: (1) obtain bacterial genomic DNA; (2) as PCR template; (3) for sequencing, genetic information analysis, etc.

Operation method

kit extraction method

Principle

This kit is designed for the purification of genomic DNA using a unique cell lysis and phase separation technique combined with the selective adsorption of DNA by a DNA preparation membrane. It is suitable for obtaining up to 20 ug of genomic DNA from 1.0x109 bacteria for molecular biology experiments such as PCR, Southern blotting analysis, RAPD, RFLD, etc.

Materials and Instruments

Bacterial Cultures
Bacterial Genomic DNA Kit
DNA preparation tubes Small volume filters Centrifuge tubes

Move

1. For first use, add anhydrous ethanol to Buffer W2 in the volume specified on the reagent bottle and mix well.
2. Prepare Buffer DV: Take 2 ml of Buffer DV-A, 125 ml of isopropanol, and 75 ml of isobutanol, add to the 250 ml reagent bottle provided and mix well.
3. Pre-cool Buffer DV at 4°C.
4. When using the kit for the first time, lysozyme with 50% glycerol to a concentration of 50 mg/ml lysozyme.
5. When using the kit for the first time, add all the RNase A carried with the kit to Buffer S and mix well.
6. Prepare a 65°C water bath.
7. Check Buffer G-A and Buffer G-B for precipitation. If precipitation occurs, heat the water bath at 65°C until the precipitate is completely dissolved before use.
8. Heat Eluent or deionized water to 65°C to facilitate the elution of genomic DNA.
Steps
1. Collect 1. ^0x109 (OD600 of 1 ml of bacterial solution is 1-1.5) of bacterial culture in 2 ml centrifuge tube, centrifuge at 12,000xg for 30 s, and discard the supernatant. Suspend the precipitate with 150 ul of Buffer S to which RNase A has been added.
~undefined Confirm that RNase A has been added to Buffer S.~undefined Suspension should be homogeneous and should not leave small clumps of bacteria, otherwise the lysis effect will be affected.
2. Add 20 ul of Lysozyme storage solution, mix well, and let stand at room temperature for 5 min.
3. Add 30 ul 0.25 M EDTA (pH 8.0), mix well, ice bath for 5 min.
4. Add 450 ul Buffer G-A, vortex and shake for 15 s, 65 ℃ water bath for 10 min.
5. Add 400 ul Buffer G-B and 1 ml Buffer DV (pre-cooled at 4°C), mix vigorously and centrifuge at 12,000xg for 2 min.
~Please prepare Buffer DV as described in Step 2 of the Experiment Preparation Procedure prior to the experiment.
6. Discard the upper phase if possible, keeping the interphase precipitate and the lower phase. Add 1 ml of pre-cooled Buffer DV at 4°C, mix vigorously, and centrifuge at 12,000xg for 2 min.
7. Discard the upper phase and transfer the lower phase to a filter (placed in a 2 ml centrifuge tube). 12,000xg for 1 min.
~The upper phase does not need to be completely discarded. Occasional mixing of the upper phase during the transfer of the colorless lower phase can be quickly phased in the Tip head and easily discarded.~If a small amount of interfacial precipitation is inhaled during the transfer, it does not need to be removed, but can be removed by filtration.~It is important to remove all colored top phases or they will inhibit the binding of DNA to the silica membrane.
8. Discard the filter and add 400 ul Buffer BV to the filtrate and mix well.
Steps 9-12 Centrifugation or negative pressure can be chosen
A. Negative Pressure
9A. Insert the preparation tube into the socket of the negative pressure device, transfer the mixture from step 8 into the preparation tube, turn on and adjust the negative pressure to -20-30 inches Hg, and aspirate the solution.
10A. Maintaining negative pressure, add 500 ul of Buffer W1 and aspirate the solution.
11A. Maintaining negative pressure, add 700 ul Buffer W2 along the perimeter of the tube wall and aspirate the solution; wash again with 700 ul Buffer W2 in the same manner.
~undefined Verify that anhydrous ethanol has been added to the Buffer W2 concentrate in the volume specified on the reagent bottle.
~undefined Adding Buffer W2 along the perimeter of the tube wall helps to thoroughly rinse the salts from the tube wall.
~Double rinsing with Buffer W2 ensures that the salts are completely removed and eliminates any effect on the digestion reaction.
12A. Place the preparation tube in a 2 ml centrifuge tube and centrifuge at 12,000xg for 1 min.
B. Centrifugation
9B. Place the preparation tube in a 2 ml centrifuge tube, transfer the mixture from step 8 to the preparation tube, and centrifuge at 12,000xg for 1 min.
10B. Discard the filtrate and return the preparation tube to the original 2 ml centrifuge tube, add 500 ul of Buffer W1 and centrifuge at 12,000xg for 1 min.
11B. Discard the filtrate and return the preparation tube to the original 2 ml centrifuge tube with 700 ul Buffer W2 at 12,000xg for 1 min.
Wash again with 700 ul Buffer W2 in the same manner.
~Confirm that anhydrous ethanol has been added to the Buffer W2 concentrate in the volume specified on the reagent bottle.
~undefined Two washes with Buffer W2 ensure that the salts are completely removed, eliminating any effect on the enzymatic reaction.
12B. Discard the filtrate and return the preparation tube to the original 2 ml centrifuge tube and centrifuge at 12,000xg for 1 min.
13. Place the preparation tube in another clean 1.5 ml centrifuge tube, add 100-200 ul of Eluent or deionized water to the center of the silica membrane, and allow to stand at room temperature for 1 min. elute the DNA by centrifugation at 12,000xg for 1 min.
~Undefined Heating deionized water or Eluent to 65°C will improve elution efficiency.

Caveat

Buffer G-B, Buffer BV, and Buffer W1 contain irritating compounds. Wear latex gloves and eyeglasses when handling and avoid contact with skin, eyes, clothing, and inhalation. If skin or eyes are contaminated, flush immediately with plenty of water or saline and seek medical advice if necessary.

Common Problems

Kit Composition, Storage, Stability

1. instructions, consumables: DNA preparation tubes, small volume filters, 2 ml centrifuge tubes, 1.5 ml centrifuge tubes.

2. RNase A: 50 mg/ml, stored at room temperature. 3.

3. Lysozyme: 50% glycerol to dissolve lysozyme, make the concentration of lysozyme 50 mg/ml, -20℃ closed storage.

4. Buffer S: bacterial protoplasm preparation solution, add RNase A, 4 ℃ airtight storage.

5. 0.25M EDTA: stored at room temperature in airtight condition. 6.

6. Buffer G-A: lysis solution, stored at room temperature under airtight conditions. 7.

7. Buffer G-B: protein removal solution, room temperature closed storage. 8.

8. Buffer DV-A: Buffer DV preparation solution, please refer to the preparation method of Buffer DV for experimental preparation, room temperature and airtight storage.

9. Buffer DV: phase separation solution, stored at room temperature in airtight container.

10. Buffer BV: DNA binding solution, stored at room temperature in a sealed container. 11.

11. Buffer W1: Wash solution, room temperature closed storage. 12.

12. Buffer W2 concentrate: desalting solution. Add anhydrous ethanol in the volume specified on the reagent bottle and mix well before use. Can be 100% ethanol or 95% ethanol.

13. Eluent: 2.5 mM Tris-HCl, pH 8.5, room temperature closed storage.

For more product details, please visit Aladdin Scientific website.

https://www.aladdinsci.com/