Isolating Genomic DNA
Genomic DNA can be isolated from:
Tail Sampling for Genomic DNA Purification
Chris Showell and Frank L. Conlon
Tail samples are taken from tadpoles at around stage 45 and later. Later stages are larger and generally provide a higher yield of tissue for DNA isolation. In addition, tail samples can be cut more posteriorly in larger tadpoles, reducing trauma and thus increasing survival. The average tissue yield is around 3 to 4mg.
To collect tail tissue samples:
Tadpoles are anaesthetized by transferring to dishes containing 0.025% ethyl 3-aminobenzoate methanesulfonate salt (tricaine/MS222; SIGMA) in distilled water. Anaesthesia usually takes 1 to 2 minutes, depending on the size of the tadpole. A scalpel is then used to cut away the posterior 1/4 to 1/3 of the tail (as measured from the posterior of the gut to the tip of the tail). A Pasteur pipette is then used to transfer the tail sample out of the dish. Tadpoles are returned to tanks containing 1 to 2 inches of water to recover. A teaspoon is a useful means of transferring the anaesthetized tadpoles. Recovery usually takes 5-10 minutes, after which the tadpoles should be able to swim and feed normally. Regeneration of the tail takes around five days. With care, a 100% survival rate can be achieved following this procedure.
Genomic DNA purification:
For genomic DNA isolation, we use a commercially available kit (Wizard SV96 Genomic DNA Purification System; Promega). Tail samples are digested overnight at 55ºC in 137.5ul of the kit’s Digestion Solution containing 1.1ug/ul proteinase K. After this point, the DNA is purified as per the kit’s ‘Animal Tissues’ protocol. The DNA yield is variable (between ~250ng and 1.0ug per mg of tissue).
Addendum by Mustafa Khokha:
The DNeasy Kit from Qiagen also works. Simply remove the tails from stage 43+ tadpoles (or the last 1/3 of the tail from early metamorphic tads) and follow the DNeasy Kit instructions. We simply use PK on the tails and do not do any further tissue disruption or homogenation.
These protocols produce genomic DNA very cheaply and can be used in a high-throughput method on samples in 48 or 96 well format. The DNA obtained is not very pure however but is adequate for PCR. Basically the tail or tadpole is lysed which yields the DNA but no further cleanup is done.
Cut tails as above. Incubate in tail lysis buffer 50 µl at 56 C for four hours in 96 well format. the store lysates at 4 C or -20.
Here's the recipe for the tail lysis buffer:
50mM Tris (pH8.8)
I keep a stock minus the proteinase K and then just add the enzyme
I've not found it necessary to do any further cleanup of the samples before PCR. The presence of the proteinase K doesn't seem to be a significant problem. I've read that heating proteinase K only reduces its activity by about 10%. I'm looking into whether it really has any effect on our Pfu. If there is any negative effect, there's a protease inhibitor (Pefabloc SC, Roche) that can be used to inactivate proteinase K, so I may try adding that after lysis.
1 M Tris (pH = 7.5)
4 M NaCl
0.5 M EDTA (pH = 7.5)
10% SDS in water
250mg/mL Proteinase K
5% Chelex 100 Molecular Biology Grade Resin (Bio-Rad #142-1253)
8-strip PCR caps (Gene Mate, ISC BioExpress)
Costar Thermowell 96 Well Polypropylene Plate
Sorvall T6000B Centrifuge (table top centrifuge with swinging buckets)
MJ Research Peltier Thermal Cycler 200
Step 1: Lysis Buffer Preparation
Calculate the volume needed for the number of embryos you plan to lyse (200µL for each embryo). Make up lysis buffer according to these specifications:
Add fresh right before use - PK to final concentration of 250mg/mL and 5% Chelex.
From our experience, it was necessary to add Chelex 100 to the lysis buffer. In our hands, samples with Chelex efficiently underwent PCR but without Chelex our PCR yields were very low to none at all.
Step 2: Creating Tad Lysis Program
Using a PCR thermal cycler, create the Tad Lysis program by setting the following temperature cycles:
1= 55ºC for 1 hour
2= 95ºC for 15 min
3= 4ºC forever
Step 3: Preparing Embryos for Lysis
Using a plastic pipette, transfer a single embryo into each well of a 96 well polypropylene plate. Use the same pipette to expel any fluid from each of the 96 wells. Add 200 µL of lysis buffer. Cap the plate with 8-strip PCR caps and place in a thermal cycler.
Step 4: Running Tad Lysis Program
Run the Tad Lysis program. After completion, the 96 well plate can be frozen until later use. Before using the collected DNA, it should be centrifuged to pellet the Chelex and lysed material. We cut our 96-well plates in half to allow them to fit in our Sorvall T6000B Centrifuge and spin them for 10 minutes. This pellets the unwanted material, allowing the DNA to be easily extracted from the supernatant.
DNA can again be isolated from either liver or spleen using the Qiagen DNeasy kit . However we recommend using half of the maximum amount of tissue recommended by Qiagen. Using higher amounts of tissue results in lower quality DNA and lower yields.
Frog should be anesthetized with benzocaine.
Or some similar buffer supplemented with proteinase K to 200 ug/ml