Author: Unkown. Date: 12/5/95
PROTEINASE K: harmful by inhalation, may cause sensitization by inhalation and skin contact.
SDS: harmful by inhalation, and if swallowed, irritating to eyes, respiratory system and skin. DO NOT BREATHE DUST!
PHENOL: poison, exposure wil burn eyes and skin. PRACTICE EXTREME CAUTION!
CHLOROFORM: highly toxic - toxic by inhalation, in contact with skin and if swallowed. Irritating to eyes, respiratory system and skin.
ETHANOL: Flammable and irritating to eyes and upper respiratory tract.
ETHIDIUM BROMIDE: powerful mutagen, may cause heritable genetic damage.
Lab coat
Gloves
Fume hood
Dust mask
For non-polymerized agarose solutions with less that 10mg/L ethidium bromide: squirt approximately 1ml bleach solution into beaker containing less than 500 ml, swirl, dispose of down the drain.
Polmerized gels containing ethidium bromide (less than 10mg/L) can be disposed of in the (non-hazardous) trash. For more information, see the UniversityÕs Òdisposal of ethidium bromideÓ policy draft in this manual.
Phenol/chloroform waste should be collected in a labeled jug, kept in the fume hood in 7-155 PWB for disposal through the hazardous waste program.
Used pipets should be placed in the fume hood until liquid is evaporated
Refer to MSDS notebook for the appropriate chemical.
Prepare Tail DNA
1. Prepare eppendorf tubes with 500 ul tail ext. solution each. Can be stored at -20.
tail extraction solution
0.2 % SDS 10% SDS 2 ml
100 mM Tris 1M Tris (pH 8.5) 10 ml
5 mM EDTA 0.5M EDTA 1 ml
500 ug/ml ProK Proteinase K 50 mg
200 mM NaCl 5M NaCl 4 ml
dH20 83 ml
100ml
Use dust mask for weighing of dry SDS
Use a 50 mg vial of Pro.K so it wonÕt be necessary to weigh out.
2. Cut 1/2 cm of the tail off using a sharp razor blade. Place tip into tube with extraction solution.
3. Place in a 55oC water bath, overnight.
4. vortex, then microfuge the tubes at high speed for 10 min.
5. transfer the supn. to a clean tube, add 1ml of Ethanol, vortex, spin for 10 min.
7. Remove alcohol and dry pellet, resuspend the pellet in 100 ul TE and store at 4oC.
PCR analysis
1. Aliqout 2 ul tail DNA (or water) into "PCR" tubes.
1. positive control DNA
2. negative control DNA
3. ...X tail DNA samples
2. Prepare a PCR "cocktail" for the appropriate number of samples.
PCR reaction cocktail
dH20 32.7-ÓYÓul times X+1 number of samples:
10X PCR buffer 5ul (for example, for 6 tails plus 2
dNTPs 8ul controls, make a 9x mixture)
MgCl Yul
primer 1 stocks are 20uM 1ul
primer 2 " " 1ul
Perkin Elmer Cetus Taq polymerase 0.3 ul
**note: add Taq last, just before adding to DNA
3. Add 48ul of the cocktail to each tube.
4. Add a drop of mineral oil on top of each sample.
5. Place samples in oiled wells of the PCR machine, run appropriate program
6. Run 30ul of each sample (plus 5ul Blue juice) on a 1% agarose gel with 5ul 1kB ladder on each side of the samples. The gel should be made with 10ul of ethidium bromide stock solution (10mg/ml) per 100 ml agarose solution. (The final concentration of ehidium bromide in the gel is 1 mg/L.) Wear gloves when pipetting ethidium bromide and handling the gel.
7. Photograph.
10X PCR buffer (1.5mM MgCl) dNTP stock (1.25mM each)
100mM Tris pH8.3 10ul EACH of 100mM stocks
500mM KCl of dATP, dGTP, dTTP, and dCTP
0.1% gelatin add 760ul dH20
15mM MgCl2 aliqout and store at -20oC
if phenol/chloroform extraction is necessary:
Gloves for the entire procedure
á Use fume hood and polypropylene tubes
á Adjust DNA solution to 500ul and .2M NaCl in water
á 1:1 Phenol/Chloroform mixture is added 1:1 with DNA/NaCl Solution
á Vortex thoroughly
á Spin 5 minutes at full speed
á Transfer aqueous phase to clean tube and extract DNA
á For example:
to 200 ml DNA + 280 ml H2O +20 ml of 5M NaCl
add 250 ml Phenol + 250 ml Chloroform