Heliconius¶
Isolating Heliconius DNA (Phenol-Chloroform-Isoamyl-Alcohol Based Protocol)
March, 18th 2019
Materials¶
Razor blades (1 per specimen); tweezers; forceps; Bunsen burner; aluminum foil and Kimwipes
2ml Phase Lock Gel QuantaBio (Heavy) (1 or more per specimen depending on how many phenol steps you will have)
1.5ml Eppendorf tubes (3 per specimen)
1 pellet pestle per sample (autoclaved)
Reagents¶
300μl of tissue digestion buffer per sample
Bleach and dH20
80% ETOH (prepare before start the extraction)
15μl proteinase K per sample
10μl RNase A per sample
Phenol: Chlorophorm: Isoamyl 25:24:1(PCI)
Chlorophorm: Isoamyl 24:1 (CI)
Ammonium Ac 5M
100% ETOH (cold)
50 - 100μl EB per sample
Preparation 1¶
Aliquot 300μl of tissue digestion buffer*1 into 1.5ml eppendorf tube (1 tube per specimen). Label both the top and side of the tube with the ID sample.
Ignite Bunsen burner to a low flame.* Remember that gloves are flammable.
Clean tweezers and forceps with dH2O, bleach (for a couple of seconds) and dH2O again. Put tweezers and forceps in ethanol 80% and flame them with ignite Bunsen burner, hold razor blade in large tweezers and heat briefly in the flame.
Use forceps and tweezers to remove parts of the butterflies (preferably thorax) from storage solution (usually DESS solution or 100% EOTH), place the piece of the butterfly onto a fresh Kimwipe. Blot away all excess ethanol or DESS solution.
Place the piece of the butterfly body on a small square of aluminum foil. Use razor blade to cut into smaller pieces. * Remember to return the rest of the butterfly to the sample tube with the storage solution. It is important that we are able to make other extractions with the same sample.
Place the “pieces” of the butterfly into appropriate eppendorf tube with 300μl of tissue digestion buffer.
Repeat steps “c” to “f” through all samples. Use a new razor blade, kimwipes, and square of aluminum foil for every sample. Flame tweezer and forceps with ethanol between samples to avoid DNA cross-contamination.
Use one pellet pestle per sample to macerate the tissues. * Needs to be autoclaved.
Preparation 2¶
Add 15μl of proteinase K to each sample tube and incubate in the shaker at 55o C, 800rpm for ~2h (until lized).
Get samples to Room temperature spin down briefly. Add 10ul RNAse A. Incubate for 10min.
Short spin down to take pieces of the eppendorf walls.
Preparation 3¶
[Preparation of tubes] Centrifuge empty Phase Lock Gel QuantaBio tube for 12,000 rpm (short button).
Take samples to the fume hood. Turn on hood fan and light. Add 1:1 volume (300μl) Phenol: Chlorophorm: Isoamyl 25:24:1 (PCI) and mix by inversion ~20 times. * PCI is highly toxic and tends to drip from the pipette, so handle with care.
Centrifuge at full speed for 10 min at Room Temperature.
In the hood, carefully remove the top layer into a new labeled 2ml Phase Lock Gel QuantaBio tube. Use P200 to remove top layer and repeat step ‘b’ and ‘c’ with ~150μl - 200 μl of PCI (repeat b and c as needed, for cleaner extract).
Remove the top layer very slowly, placing the tip in the top of the layer in order to not get anything from the bottom layer.
Place the eppendorf in a vertical position.
Always leave a lit bit of the top layer trying to avoid to get anything from the bottom.
Discard the bottom layer liquid in a discard bottle labeled “phenol: chloroform: isoamyl alcohol + Name of project + Barrett Lab” that is placed in the hood and discard the tubes in the container also in the hood.
Separate upper phase into new 1.5ml eppendorf tube. Add 1:1 volume of Chlorophorm: Isoamyl 24:1 (CI) and mix by inversion ~20 times (same volume of upper phase and CI).
Use regular eppendorf. DON’T USE SAFE LOCK GEL TUBES with CI.
Centrifuge at full speed for 10 min at Room temperature.
Carefully remove the top layer into a new 1.5ml Eppendorf tube. Add 1:10 volume of Ammonium Ac 5M (one tenth of the volume of the upper phase) and then 2 volumes of 100% ETOH (cold). Mix by inversion ~20 times.
For example, if you recover 300μl of the upper phase, you add 30μl Ammonium Ac and 600μl of 100% ETOH.
Discard the bottom layer liquid in a discard bottle labeled “chloroform: isoamyl alcohol + Name of project + Barrett Lab” that is placed in the hood and discard the tubes in the container also in the hood.
Incubate at -20 oC for ~15min.
Centrifuge at 4 oC for 15 min. Decant supernatant (better leave a little bit of ETOH)
If you decant the supernatant you are removing the Ammonium Ac, ETOH and the remaining of the extraction that is not DNA. Be careful not to remove the pellet. In this step it is better to leave a little bit of the supernatant.
Wash pellet with 300μl of 80% ETOH (cold) per sample, mix by flicking “with fingers” or shaker 800 rpm.
for 6 samples prepare 5400ul of 80% ETOH (4,320ul of 100% EOTH + 1,080ul of ddH2O)
Centrifuge at 4 oC for 15 min. Decant supernatant.
Dry pellet at Room temperature.
Elute in 50-100μl Elution Buffer (EB) 🡪 Doing with 60 μl
Note
For cleaner extraction, you can follow with a 2X SPRI bead wash, this can be repeated as needed, with some DNA loss.
Use wide bore tips whenever possible and minimize pipetting for higher Molecular Weight DNA.
Store DNA at 4 oC and keep away from light.
Solutions¶
[C1V1 = C2V2]
Tissue digestion buffer STRI
60mM TRIS-HCl pH 8
100mM EDTA pH 8
0.5% SDS
Combine the following to make 500 ml:
345ml of ddH2O
30ml of 1 M Tris-HCl pH 8.0
100ml of 0.5 M EDTA
25ml of 10% SDS
Proteinase K (20 mg/ml, comes in solution)
RNase A (100mg/ml, comes in solution)
25:24:1 Phenol: chloroform: isoamyl alcohol solution (comes in solution)
100% ethanol (100mg/ml, comes in solution)
80% ethanol
Combine the following to make 10ml:
8ml of 100% ETOH
2ml of ddH2O
Combine the following to make 50ml:
40ml of 100% ETOH
10ml of ddH2O
EB: 10 mM Tris-HCl pH 8.0
Combine the following to make 250ml
2.5ml of Tris-HCl 1M pH 8 (same as 2500μl)
247.5ml of dH2O (same as 247ml + 500μl)
DESS Solution: 0.5M EDTA pH 8 + DMSO 20% + NaCl
Combine the following to make 500ml
250ml of 0.5M EDTA pH 8
100ml DMSO (final concentration 20%)
105g NaCl
400 to 450ml of ddH2O