PCR-Products and GEL/DNA Purification Kit
Gel Extraction/DNA Purification Kit
Cat No: PPG-50/ PPG-100 / PPG-200
Introduction:
- Gel Extraction/DNA Purification Kit is a rapid and reproducible method for the purification and concentrating of high-quality, double-stranded DNA from enzymatic reactions such as PCR, restriction digestion, ligation and reverse
- The protocol involves binding, washing and elution workflow with minimal incubation and spin times, thus resulting in purification in less than 5
- The washing step with Wash Buffer results in removal of enzymes, short primers, detergents and other low-molecular weight reaction components or impurities, thereby allowing low-volume elution of concentrated and high-purity DNA for future use.
- Eluted DNA is ready for use in other enzymatic
- The protocol can also be undertaken in order to enable the purification of smaller DNA fragments,
Description
Materials Provided:
| Component | 100 Preps | 5 Preps |
| *DNA Cleanup Binding Buffer | 40 ml (add 20 ml of 96-
100% Isopropanol) |
2 ml (add 1 ml of 96-100%
Isopropanol) |
| **DNA Wash Buffer | 10 ml (add 40 ml of 96-
100% ethanol) |
0.5 ml (add 2 ml of 96-
100% ethanol) |
| DNA Elution Buffer | 10ml | 1 ml |
| Mini Columns | 100 | 5 |
| Tubes as column inserts | 100 | 5 |
Additional requirements: microcentrifige Tubes , (96-100%) absolute ethanol , (96-100%) isopropanol
Note:
*Add 96-100% isopropanol to DNA Cleanup Binding buffer as written on the bottle before using.
**Add 96-100% of ethanol to DNA Wash buffer as written on the bottle before using.
*You can use either Isopropanol or Ethanol in binding buffer but preference should be given to Isopropanol as it gives better results.
Procedure:
- Add 95% ethanol to DNA Wash Buffer prior to use in the ratio of 4: 1 (i.e. 4 volumes of 95% ethanol and one volume of DNA Wash
- Dilute sample with DNA Cleanup Binding Buffer in specific ratio of sample: binding buffer depending the size of the
For example:
ssDNA (cDNA), sample to binding buffer ratio 1:6 dsDNA (< 2.5 kb), sample to binding buffer ratio 1:5 dsDNA (> 1.5 kb), sample to binding buffer ratio 1:2
For purifying DNA from gel slice:
Take 25 to 50 mg of gel slice into a clean microfuge tube. Add 500 ul binding buffer (containing Isopropanol) and warm the tube at 55°C for 15 minutes or more until the gel slice dissolves. Proceed to the next step – loading directly into the spin column.
- Mix well by pipetting up and down or by tapping the Do not vortex.
- Insert column into collection tube and load upto 600 μl sample into the column. Spin for 1 minute at 16,000 x g or at maximum speed in micro centrifuge. Discard the flow-through. Repeat this step if volume is more than 600 μl.
- Re-insert column into collection Add 250 μl of DNA Wash Buffer and spin for 1 minute at maximum
- Transfer column to a clean 5 ml microfuge tube and spin for 1 minute to ensure the removal of traces of salt and ethanol, so that they are not carried over to next step. Discard the microfuge tube and save the column for next step
- Place the Column in a clean 5 ml microfuge tube and add 10–20 μl of DNA Elution Buffer to the center of the column. In order to obtain maximum elution, make sure the Elution buffer is in direct contact with membrane in the center rather than sticking to the wall of the column.
- Wait for 1 minute, and then spin at maximum speed for 1 minute to elute
Note: Nuclease-free water can also be used to elute the DNA. If a larger volume of DNA Elution Buffer is used, yield may slightly increase, however, this would result in the dilution of DNA sample. Hence, typical elution volume should stay 6–20 μl. For larger size DNA (≥ 10 kb), heating the elution buffer to 50 °C prior to use can improve yield.
- Visualize the eluted PCR products or DNA by loading on to agarose gel
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