Week 08 DNA extraction
Week 08 DNA extraction kdorfman Mon, 07/24/2023 - 20:25Dog DNA labs, week of 10/23/23
Isolate DNA
1 sample per pair, from:
- Dog cell cultures from Kathleen
- Student dog mouth swabs
- DNA from Cornell BioBank
- Staff dog mouth swabs
QIAamp DNA minikit 51306
Equipment
- Microfuge tubes
- Sterile forceps (to pull swab from extraction buffer)
- QIAamp columns
- collection tubes
- 56C heat block (10 min)
- vortex mixers
- centrifuge 6000 x g 1 min, 20K x g 3 min
- Nanodrop (may need next class, also) (Need to calculate uL to get 100 ng)
Reagents (per swab)
- PBS (400 UL)
- Proteinase K (20 uL)
- Buffer AL (400 uL)
- 95% EtOH (400 uL)
- QIAamp Mini spin column (1)
- 2 mL collection tube (2?)
- Buffer AW1 (500 uL)
- Buffer AW2 (500 uL)
- Buffer AE (50 uL)
Staff makes spreadsheet on Google Drive with:
- sample # for each dog
- concentration
- quality
DNA from cultured cells
DNA from cultured cells kdorfman Tue, 08/22/2023 - 16:04From QIAamp DNA Mini & Blood Mini Handbook, Apendix B, p 50
Do not use more than 5 x 10^6 cells
Important points before starting
- Do not use more than 5 x 106 cells (with a normal set of chromosomes).
- All centrifugation steps are carried out at room temperature (15–25°C).
- Use carrier DNA if the sample contains <10,000 genome equivalents (see page 17).
Things to do before starting
- Heat a water bath or heating block to 56°C.
- Equilibrate Buffer AE or distilled water to room temperature (15–25°C) for elution.
- Ensure that Buffer AW1, Buffer AW2, and QIAGEN Protease have been prepared according to the instructions on page 16.
- If a precipitate has formed in Buffer AL, dissolve by incubating at 56°C.
Procedure
- Harvest cells
- Trypsinize and count cells
- Put no more than 5 million cells into a microfuge tube
- Centrifuge for 5 min at 300 x g.
- Remove the supernatant completely and discard, taking care not to disturb the cell pellet.
- Resuspend cell pellet in PBS to a final volume of 200μL.
- Add 20 μL QIAGEN Protease or proteinase K.
- (Continue with step 3 of “Protocol: DNA Purification from Blood or Body Fluids (Spin Protocol)”, page 27)