Description
Materials Provided:
Material | 100 Prep | 5 Prep |
*Resuspension Buffer | 30 ml | 1.5 ml (RNase A added) |
Rnase A Solution (10 µg/µl) | 300 µl | – |
Lysis Buffer | 30 ml | 1.5 ml |
Neutralization Buffer | 30 ml | 1.5 ml |
DNA Binding Buffer (Brown Bottle) | 40 ml | 2 ml |
**Wash Buffer | 20 ml (add 80 ml of
96 -100 % ethanol |
1 ml (add 4 ml of 96 –
100 % ethanol |
Elution Buffer | 20 ml | 1 ml |
Mini Columns | 100 | 5 |
Tubes as column inserts | 100 | 5 |
Additional requirements: Microcentrifige Tubes , 96 – 100% Absolute ethanol.
Note: **To Wash Buffer add 96-100% ethanol (In 20 ml of wash buffer + 80 ml ethanol).
*Add Rnase A Solution 300 µl to 30 ml Resuspension Buffer.
Procedure:
- Pipette about 1 ml of coli cells into a 1.5 ml microfuge/Eppendorf tubes. Centrifuge the sample at 10,000 rpm for 2-5 minutes at room temperature.
- Discard the supernatant, and resuspend the cell pellet in 250 µl of Resuspension Buffer containing RNase A . Mix by tapping
- Add 250 µl of Lysis Buffer to the cell (Do not vortex)
- Mix the suspension by gently tapping or by inverting the tube up and down 8-10
- Add 250 µl of Neutralization Buffer and mix the solution thoroughly by inverting the tube up and down 8-10 (Do not vortex).
- Centrifuge at 10,000-14,000 rpm for 10 Discard the pellet and save the supernatant.
- Add 375 µl of DNABinding Buffer to the clear supernatant and
- Load 550-600 µl of the mixture on to the DNA spin column, centrifuge for 1 -2 minutes and discard the flow
Note: You can save the remaining half of the lysate and freeze it at -20°C for future use. If you plan to use all of it now, this will probably double the amount of the DNA yield.
- Wash the DNA spin column with 400 µl of Wash Centrifuge the column for 1-2 minutes. Discard the flow through. Wash one more time.
Reviews
There are no reviews yet.