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Isolating Genomic DNA

Genomic DNA can be isolated from:

  1. Tadpoles - using kits
  2. Tadpoles - without kits cheap but dirty
    1. Showell lysis buffer (Conlon lab)
    2. Schmidt-Grammer protocol (Harland Lab)
  3. Frogs/Froglets
  4. Nucleated RBC's (lots and in high quality)

Tadpoles - Using Qiagen or Wizard

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.

Tadpoles - DNA purification without kits

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.

Protocol By Chris Showell (Conlon Lab)

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)
1mM EDTA
0.5% Tween 20
200ug/ml proteinase K (PCR grade)

I keep a stock minus the proteinase K and then just add the enzyme
before use.

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.

Protocol by Philip Schmidt and Tim Grammer (Harland Lab)

Pre-collection Checklist:

Reagents:

1 M Tris (pH = 7.5)

4 M NaCl

0.5 M EDTA (pH = 7.5)

10% SDS in water

ddH2O

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

 

Instruments:

P200 pipettor

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:

                   

Total Volume (mL) 10 Final Concentration
Tris (pH = 7.5) 1M 0.5 50mM
NaCl 5 M 0.125 50mM
EDTA 0.5 M 0.1 5mM
SDS 10% 0.5 0.5%
ddH2O 8.775

                   

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.

 

Frogs/Froglets (Mustafa Khokha)

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.

DNA can also be isolated from the nucleated RBC's in large amounts: (Dan Roche and Mustafa Khokha)

Frog should be anesthetized with benzocaine.

Open the ventral surface to expose the heart, preferably with a small hole so the liver does not pop out. Cut the cartilaginous shield-shaped bone connecting the shoulders with a heavy scissors and remove. The heart should be visible. Carefully lift the silver pericardium away from the heart and cut open to expose the heart. Invert the frog so the heart is in contact with a 2059 tube (15 ml polypropylene – filled with 0.85x SSC). With a pair of small scissors, nick the aorta and submerge into the .85x SSC solution. The heart will pump the circulating blood into the collection tube. Once all the blood is collected, cap the tube and invert to prevent clumping of the RBCs.

Spin down at 2,000 rpm for 5 min.

Decant off the supernatant, then resuspend the nuclei in 0.85x SSC to a volume equivalent of 100-200ug/ml DNA in a 50 ml conical tube (100ug DNA ~ 3.2x107 cells). Cell numbers can be counted with a hemacytometer, or we typically resuspend in 10 ml of 0.85xSSC. Ideally the resuspension should be complete to avoid lumps.

Add an equal volume of :

1% SDS
20 mM EDTA
100 mM NaCl
20 mM Tris pH 7.5-8.0

Or some similar buffer supplemented with proteinase K to 200 ug/ml
Incubate overnight at 55°C (some use 37°C)

It is important that the solution not be lumpy. (Never vortex genomic DNA)

Perform a phenol extraction. Add an equal amount of buffered phenol to lysis buffer. Use an end over end or equivalent mixing device to keep the two phases mixing. Mix for at least an hour.

Spin at 2,000 rpm for 5 min.

Add 1/4 vol 10M NH4Acetate, mix thoroughly, then add 0.6 volumnes isopropanol and mix the viscous solution thoroughly. The DNA should be visible and condense into a large stringy lump. Spool the DNA on a clean Pasteur pipet (which has been closed by flaming).. Scrape the DNA into a clean tube of 70% EtOH.

Spool the DNA again, with a clean Pasteur pipet (end closed), place in a clean tube. Remove all traces of EtOH.

Resuspend in about 1-2 ml of TE pH 8.0 (Resuspension may take several hours. Agitate on a rotator or similar device. Measure the OD).

Should be ready for digestion.