BBL Gel Extraction Kit; Spin Column

Cat# BB-GEK50 (50 preps)

Introduction:

BBL-Gel Extraction Kit is a column based nucleic acid purification kit where the column has silica membrane which is capable of purifying DNA fragments easily from TAE or TBE agarose gel (both normal and low melting point agarose) with significant removal of proteins and other organic impurities utilizing the optimized buffer system. 

This kit can purify DNA fragments of 80 bp to 10Kbp with a recovery rate of more than 75%. The purified DNA is suitable for basic molecular biology experiments like restriction digestion, ligation, PCR amplification etc.

Kit Contents:

Storage:

BBL Gel Extraction Kit (# BB-GEK50) is stable for 12 months if stored properly in dry place at room temperature (20°C – 25°C) not exposed to sunlight.

Note:

• Add ethanol (96-100%) to Wash buffer before use (see bottle label for volume)
• All centrifugation steps are carried out at 12000 rpm (~13400 x g) in a conventional table top centrifuge at room temperature (15-25°C).
• Ensure that the elution buffer is dispensed directly onto the membrane for complete elution of bound DNA. The average volume of elution buffer is ~50 µl. Less volume of elution buffer may leads to lower recoveries of DNA.

Protocol

1. Excise the desired DNA fragment from the agarose gel with a clean, sharp scalpel.

Remove extra agarose to minimize the gel slice size.

High wavelength UV & short exposure time of gel during excision will reduce UV-induced mutation.

2. Weigh the gel slice in a clean colourless micro-centrifuge tube. Add 1 gel volume of Binding Buffer to the tube. (100 mg ~ 100 µl).

3. Incubate at 55° for 5-10 minutes (or until the gel slice dissolves completely). Mix by inverting tube intermittently to ensure dissolving the gel.

For >2% gels, increase the incubation time.

NOTE: If the target DNA fragment size is <300 bp, add ½ gel volume of Isopropanal to the sample and mix

4. Place a spin column in a 2 ml collection tube provided in the kit.

5. To bind DNA, apply the sample to the spin column. Keep at room temperature for 2 min for binding and centrifuge for 1 min

The maximum volume of the column reservoir is 750 µl. For sample volumes of more than 750 µl, repeat the step 5 again.

6. Discard the flow through and place the spin column back in the same collection tube.

7. Add 600 µl of Wash Buffer to the column and centrifuge for 1 min. Discard the flow through and place the spin column into the collection tube.

8. Repeat the above step 7 for one more time.

9. Discard the flow through and centrifuge the tube for an additional 1 min

Residual ethanol from Wash Buffer will not be completely removed unless the flow through is discarded before this additional centrifugation.

10. Place the spin column into a fresh, clean 1.5 ml micro centrifuge tube.

11. To elute DNA, add 50 µl of Elution Buffer to the centre of the membrane. Incubate at room temperature for 3-5 minutes and then centrifuge for 2 min.

12. Take 2-5 µl purified DNA for concentration check and electrophoresis and stored the rest of the DNA at -20°C for further analysis.