I’ve been trying to extract C. elegans genome for WGS over the past 5 months and every time I do I either get mainly degraded DNA or sometimes perfect DNA. I only get perfect DNA once in every 20 preps. I use heat of 56C for 1.5 hrs in the T1 buffer from the machnery-nagel DNA Tissue kit along with proteinase K, followed by RNase I treatment at 1 mg/ml for 10 minutes at room temperature then a standard ethanol precipitation. (I would love to say exactly what is in the T1 buffer but I cannot as it is a company secret) Does anyone have a new method for genome extraction as I find this one doesn’t lead to good results, preferably one that doesn’t involve heat. Or even how to quickly dissolve the worm that doesn’t affect the DNA?
I have also tried leaving the worms in heat for longer at 42C but this results in fully degraded. Also I have done beads, multiple freeze thaw cycles, and adding BME to the lysis buffer. Nothing is working.
Hi there, I have had great results using Qiagen’s DNeasy kit. Unfortunately, it does use heat as it utilizes proteinase K for digestion, but I have never had noticeable problems with degradation.
I’ve generally been happy with a modified version of the Michael Koelle protocol for Southern Blots - extremely old fashioned, it involves proteinase K digestion followed by phenol/chloroform and spooling of the precipitated DNA (rather than centrifugation). But it involves proteinase K and so heating, which you don’t want.
There are other options for getting at worm genomic DNA, that don’t involve heating. You can look for reports of using a Balch Homogenizer or a so-called “bead beater” to extract nuclei from the otherwise tough worm. This report of using a Balch Homogenizer, for example, stresses that its use avoids freezing and heating.
Hello,
I have used the following protocol for several years with excellent results (yields of over 100ug diluted in 50uL, running in a single band of >15kb on a 1% agarose gel). I hope this helps!
WORM GENOMIC DNA PURIFICATION FOR WHOLE-GENOME SEQUENCING
a) Growing and collecting worms
- Chunk worms onto 16 plates.
- Wash off worms with M9 after the plates are close to starvation (or they are slightly starved) and harvest
them into Falcon tubes (8 plates per tube). - Spin it down, remove supernatant, and add M9 medium.
a. Repeat this step 3 times. - Leave them resting for 2 hours in M9 at RT on a mixer/nutator in order to get rid of the bacteria in the gut.
- Rinse with M9 medium as above 3 times and move to a 1.5mL microfuge tube with glass pipette.
a. The goal is to get ~100μl of packed worms.
b. Worms can be stored at -80°C for a long time before prepping them.
b) Preparing worms
- Thaw a 100μl pellet and add (in the hood):
- 350 μl of Lysis buffer
- 12 μl of 20% SDS (20g in 100ml of H20 )
- 2 μl of 20mg/ml proteinase K in H20
- 1 μl of β-mercaptoethanol
- Mix it by inverting, wrap the tube with parafilm, and incubate it at 65 °C (water bath) for 1 hour.* Do NOT
incubate longer than that or increase the temperature, as that would lead to the degradation of the gDNA.
*Hammarlund lab protocol: 1-2 hours (up to 3) at 55°C.
c) DNA extraction and purification
-
Perform a double Phenol/CHCl3 (Invitrogen, Ref. 15593-031) (stored at 4°C) extraction followed by a
chloroform extraction:
a. Add 235 μl of Lysis buffer to increase the final volume up to 700μl.
b. Add 1 volume of Phenol/CHCl3 (700μl) under the hood.
c. Mix gently by hand inverting (35 times), making sure that both phases make contact. Wait for 1
min until both phases are defined.
d. Spin down for 5 min at max speed at RT.
e. Collect 500μl of supernatant and transfer it into a new tube. Be very careful while removing the
upper phase to avoid disturbing the interface.
f. Add 200 μl EB Buffer (to increase volume back to 700μl) and then 1 volume of Phenol/CHCl 3
(700μl) under the hood.
g. Mix gently by hand inverting (35 times), making sure that both phases make contact. Wait for 1
min until both phases are defined.
h. Spin down for 5 min at max speed at RT.
i. Collect 600μl of supernatant and transfer it into a new tube. Be very careful while removing the
upper phase to avoid disturbing the interface.
j. Add 1 volume of pure chloroform (CHCl3 ) (600μl) under the hood.
k. Mix gently by hand inverting (35 times), making sure that both phases make contact. Wait for 1
min until both phases are defined.
l. Spin down for 5 min at max speed at RT. -
Collect 400μl of supernatant and transfer it into a new tube. Be very careful while removing the upper phase to avoid disturbing the interface.
-
Perform Ethanol/NaOAc precipitation as follows:
a. Add NaOAc (3M, pH 5.2) at 1:10 ratio (in this case, add 40μl, since the initial volume is 400μl).
b. Add 2.5 volumes of 100% EtOHΨ (in this case, add 1000μl, since the initial volume is 400μl).
*Chill the EtOH solution by keeping it on ice while you are working. -
Mix gently by hand inverting (35 times). A white, mucus-like mass is generated, reflecting the
precipitation of both nucleic acids and salts. Incubate the solution in freezer for 30 min. -
Spin down for 30 min (at max speed) at 4°C (centrifuge stored in the cold room). Mark the side of the tube
where the pellet is expected to be. -
Remove the supernatant and add 400μl of EB buffer. Wrap the tube up with parafilm and incubate it at
37 °C (water bath) for 15 min to dissolve the DNA. -
Add RNAse at 4mg/ml (add 2.30μl of RNAseA solution in 400μl). Wrap the tube with parafilm and
incubate it at 37°C (water bath) for 30 min. -
Perform a double Phenol/CHCl 3 extraction followed by a chloroform extraction:
a. Add 300 μl of EB buffer to increase the final volume up to 700μl.
b. Add 1 volume of Phenol/CHCl 3 (700μl) under the hood.
c. Mix gently by hand inverting (35 times), making sure that both phases make contact. Wait for 1
min until both phases are defined.
d. Spin down for 5 min at max speed at RT.
e. Collect 500μl of supernatant and transfer it into a new tube. Be very careful while removing the
upper phase to avoid disturbing the interface.
f. Add 200μl EB Buffer (to increase volume back to 700μl) and then 1 volume of Phenol/CHCl3
(700μl) under the hood.
g. Mix gently by hand inverting (35 times), making sure that both phases make contact. Wait for 1
min until both phases are defined.
h. Spin down for 5 min at max speed at RT.
i. Collect 600μl of supernatant and transfer it into a new tube. Be very careful while removing the
upper phase to avoid disturbing the interface.
j. Add 1 volume of pure chloroform (CHCl 3 ) (6 00 μl) under the hood.
k. Mix gently by hand inverting (35 times), making sure that both phases make contact. Wait for 1
min until both phases are defined.
l. Spin down for 5 min at max speed at RT. -
Collect 400 μl of supernatant and transfer it into a new tube. Be very careful while removing the upper
phase to avoid disturbing the interface. -
Perform Ethanol/NaOAc precipitation as follows:
a. Add NaOAc (3M, pH 5.2) at 1:10 ratio (in this case, add 40 μl, since the initial volume is 400μl).
b. Add 2.5 volumes of 100% EtOHΨ (in this case, add 1000 μl, since the initial volume is 400μl).
*Chill the EtOH solution by keeping it on ice while you are working. -
Mix gently by hand inverting (35 times). A white, mucus-like mass is generated, reflecting the
precipitation of both nucleic acids and salts. Incubate the solution in freezer for 30 min. -
Spin down for 3 0 min (at max speed) at 4°C. Mark the side of the tube where the pellet is expected to be.
-
Remove the supernatant and wash the pellet with 70% EtOH* to remove salts as follows:
*Chill the 70% EtOH by storing it on ice while you are working.
a. Add 400μl without disturbing the pellet.
b. Wash the pellet by gently inverting the tube several times (~35 times).
c. Spin it down for 5 min (at max speed) at 4°C (centrifuge stored in the cold room).
d. Perform the washing step 3 times. -
Remove the supernatant and air-dry the pellet by leaving it open under the hood for 10 min.
-
Add 50μl of nuclease-free water.
-
Seal the tube with parafilm and store it at 37°C overnight (in an incubator).
d) Measuring the concentration of DNA and quality control
-
Measure the DNA concentration by using NanoDrop Spectrophotometer Stranded DNA setting (use
nuclease-free water for blanking). The concentration must be greater than 20ng/μl and the total DNA mass
must be greater than 1.5μg. -
Check the quality of your gDNA sample.
a. Measure the 260/280 ratio by using NanoDrop Spectrophotometer Stranded DNA setting. The
ratio of absorbance at 260nm and 280nm provided by the NanoDrop Spectrophotometer is used to
assess the purity of DNA and RNA. A ratio of ~1.8 is generally accepted as “pure” DNA; a ratio of
~2.0 is generally accepted as “pure” RNA. If the ratio is appreciably lower in either case, it may
indicate the presence of protein, phenol or other contaminants which absorb strongly at or near
280nm.
b. Measure the 260/230 ratio by using NanoDrop Spectrophotometer Stranded DNA setting. The
260nm/230nm ratio is used as a secondary measure of nucleic acid purity. The 260/230 values for
“pure” nucleic acid are often higher than the respective 260/280 values. Expected 260/230 values are
in the range of 2.0-2.2. If the ratio is appreciably lower than expected, it may indicate the presence of
contaminants which absorb at 230nm, such as phenol, TRIzol or even carbohydrates, which have
absorbance near 230nm.
c. Run 5 μl of the sample (add 1- 2 μl of the loading dye and load it all) against 6 - 7 μl of 1kb DNA
ladder. Image the gel.
d. You should obtain a single band >10kb. A lower well-defined band might be reflecting RNA or
bacteria gDNA contamination. A diffuse pattern might be indicating degraded gDNA. Label and
save the gel picture. -
Store your gDNA at - 80 °C.
Thank you for the feedback.
I will look into the Qiagen’s DNeasy kit for sure!
As for the southern blots protocol I did already try that with the BME and SDS in the lysis buffer but that lead to my worst degradation yet.
I was able to extract perfect DNA once last week on samples that didn’t matter but now I fail to replicate the process.
As for the Worm genomic protocol I will give this a try and see if it helps.
Thank you again for all of your help!