1. PGA DNA Extraction V2.1
2. Fluorescent Genotyping PCR V2.1
3. PCR Mix Macro (associated with the PCR)
4. Pooling and Purifying V2.1
5. Creating Sample Sheets V2.1
6. Sample Sheet Macro (associated with creating sample sheets)
Kathleen L. Kennedy
With
Howard J. Jacob, Ph.D.
Revised 11/19/02
PGA DNA Extraction and Spec Protocol
Recipes:
Lysis Buffer (200ml)
Sterile autoclaved dH2O
158ml
2mM Tris (pH 8.5)
10ml
0.05M EDTA (pH 8.0)
20ml
10% SDS
4ml
5M NaCl
8ml
For every ml of lysis buffer to be used add 100ug of Proteinase K.
This translates into 10ul of 10mg/ml PK for every 1ml of active lysis buffer that you
intend to use.
Do not activate your entire lysis buffer unless you intend to use it all that same day!
Resuspension Buffer (200ml)
Sterile autoclaved dH2O
198.6ml
2M Tris (pH 7.5)
1ml
0.05M EDTA
400ul
Tissue Information
Approximate size: ear punch (1mm diameter) or tail (1-2mm piece)
Approximate weight:
Procedure:
1. Add 500ul of lysis buffer to each tube.
- Lysis buffer should be made fresh each time used
- To calculate the total volume needed count all the tubes and divide by two
- Measure out the buffer in a 50ml tube and add Protein Kinase (10% of the
volume of buffer) e.g. If 25ml lysis buffer then add 250ul of protein kinase
- Mix the solution gently to prevent the formation of bubbles
2. Shake the tubes to ensure that the tissue is in solution.
- Invert the tubes to be sure that the tissue is visible through the cap of the
tube
3. The tubes are placed in a 55º C water bath with agitation over night.
- Orient the so the cap of the tube is angled downwards
4. Remove the tubes from the water bath the next morning and allow to reach room
temperature (about 30 minutes)
5. Add 500ul of 100% Isopropyl to each tube to precipitate the DNA.
6. Shake the tube vigorously for 3 minutes.
7. Place the tubes in the centrifuge for 15 minutes at 14000 rpm
- Remove immediately
8. Pour off the liquid being sure not to lose the pellet.
- Do not recap the tubes.
9. Add 500ul of 70% ethanol to each tube, cap, and shake.
- This is done to clean the DNA and remove salts
10. Place the tubes in the centrifuge for 5 minutes at 14000rpm.
11. Pour off the liquid being careful not to lose the pellet.
12. Leave the tubes uncapped for a minimum of 1 hour to allow any ethanol to
evaporate.
K. L. Kennedy
2
13. Resuspend the DNA in 500ul of re-suspension buffer.
14. Shake the tubes vigorously and place in a 55C water bath for the weekend, or a
minimum of overnight.
- The tube should now be placed with the cap facing upward.
- To aid in resuspension the tube should be shaken vigorously after being in
the water bath for 1 hour
15. The samples are now ready for reading on a spectrophotometer. We do a 1:20
dilution for the DNA spec. We dilute samples in water for use.
K. L. Kennedy
3