Tribolium castaneum DNA extraction, Ximena Gutierrez Ramos & Patricia Graham
Beetle book: genomic DNA extraction protocol with modifications
Solutions:
Homogenization buffer - 80 mM EDTA pH8, 100 mM Tris pH 8 and 0.5% SDS (autoclave).
Proteinase K
RNAse
Phenol equilibrated 0.5 M Tris pH 8
Chloroform
Ethanol 100% and 70%
3M sodium acetate buffer
genomic DNA extraction protocol (based in Drosophila DNA extraction protocol)
Solutions:
Buffer A: 100 mM Tris-HCl pH 7.5, 100 mM EDTA, 100 mM NaCl and 0.5% SDS. Store at RT.
Buffer B: 5M potassium acetate and 6M lithium chloride.
Isopropanol.
Ethanol 70%.
Leg squish: genomic DNA extraction protocol
Solutions:
Squishing buffer: 100 mM Tris-HCl pH 8.2, 1 mM EDTA, 25 mM NaCl and 200 ug/uL proteinase K.
Note: We use the leg squish protocol for screening lines by PCR. In most of the cases, once we remove the leg we separate the adult beetle in an individual vial until we know the PCR result.
Beetle book: genomic DNA extraction protocol with modifications
Solutions:
Homogenization buffer - 80 mM EDTA pH8, 100 mM Tris pH 8 and 0.5% SDS (autoclave).
Proteinase K
RNAse
Phenol equilibrated 0.5 M Tris pH 8
Chloroform
Ethanol 100% and 70%
3M sodium acetate buffer
- Place 15 pupae with 250 uL Hom buffer in a 1.5 mL tube and homogenize with a pestle, then add 750 uL Hom buffer homogenize the sample with the pestle. The sample is going to be slimy.
- Add Proteinase K to a final concentration of 100 ug/mL.
- Incubate 2 h at 50C, rock from time to time.
- Centrifuge 10 min at 13 000 rpm and transfer the supernatant to a new tube. Be careful to not take parts of the beetle, if that happens centrifuge again!
- Incubate 30 min at 80C to inactivate Proteinase K.
- Add 500 uL phenol and 500 uL chloroform.
- Vortex 1 to 2 min.
- Centrifuge 10 min at 13 000 rpm and transfer the aqueous phase to a new tube. Be careful, don’t take the interfase!
- Repeat steps 6 to 8 for 3 times. You are going to see that the aqueous phase is a little less pigmented than the first time of the phenol:chloroform wash and you are not going to have an interphase the third time.
- Add 500 uL chloroform, vortex and centrifuge 10 min at 13 000 rpm.
- Transfer the upper phase into a new tube. Add 1/10 volume of 3 M sodium acetate pH 6 and add 2 volumes of ethanol 100%. Invert to mix and incubate ON -20C.
- The next day centrifuge 10 min at 13 000 rpm, remove the solutions to have only the pellet.
- Wash the pellet with 70% ethanol, centrifuge 10 min at 13 000 rpm.
- Remove the ethanol and let the ethanol evaporate at RT for about 10 min.
- Resuspend with 50 uL water.
genomic DNA extraction protocol (based in Drosophila DNA extraction protocol)
Solutions:
Buffer A: 100 mM Tris-HCl pH 7.5, 100 mM EDTA, 100 mM NaCl and 0.5% SDS. Store at RT.
Buffer B: 5M potassium acetate and 6M lithium chloride.
Isopropanol.
Ethanol 70%.
- Collect 15-20 adult beetles in a 1.5 mL tube placed on ice.
- Grind the beetles in 200 uL Buffer A with a disposable tissue grinder. Add an additional 200 uL Buffer A (total volume 400 uL Buffer A). Continue grinding until you can observe only the cuticle.
- Incubate samples at 65C for 30 min.
- Add 200 uL 5M potassium acetate and 500 uL 6M lithium chloride, mix well by inverting the tube (6 to 10 times). Incubate on ice for at least 10 min and up to a few hours.
- Centrifuge 15 min at 13 000 rpm and transfer the supernatant to a new tube. Be careful to not take parts of the beetle, if that happens centrifuge again!
- Add 600 uL isopropanol and mix by inverting the tube.
- Centrifuge 10 min at 13 000 rpm.
- Discard the supernatant. Wash the pellet with 70% ethanol, centrifuge 10 min at 13 000 rpm.
- Remove the ethanol and let the ethanol evaporate at RT for about 10 min.
- Resuspend with 20-50 uL water.
Leg squish: genomic DNA extraction protocol
Solutions:
Squishing buffer: 100 mM Tris-HCl pH 8.2, 1 mM EDTA, 25 mM NaCl and 200 ug/uL proteinase K.
Note: We use the leg squish protocol for screening lines by PCR. In most of the cases, once we remove the leg we separate the adult beetle in an individual vial until we know the PCR result.
- Prepare the mL of squishing buffer necessary for the beetles that you are going to squish. We usually used 40 uL of one leg.
- Sift the beetles from the flour, put the beetles to sleep with CO2 until they stop moving. Move them on their dorso and remove 1 of the legs of the second pair of legs with a forceps.
- Put the leg in a PCR tube, squish the leg with a yellow tip loaded with 20 uL of Squishing buffer. You can observe that the squishing buffer helps the grinding process of the leg. Add 20 uL of squishing buffer (40 uL total). Put on ice until you finish all the samples.
- Return the beetle to a new vial with flour.
- Incubate the leg samples 30 min at 37C and 2 min 95C. Store legs at -20C.
- For the PCRs we used 1 to 2 uL of each leg squish sample.