Aug 07, 2017
Version 4
High quality DNA from Fusarium oxysporum conidia suitable for library preparation and long read sequencing with PacBio V.4
- Sukhwinder Kaur1,
- Quyen Anh Pham1,
- and Lynn Epstein1
- 1Dept. of Plant Pathology, University of California, Davis, CA, 95616 USA
Protocol Citation: Sukhwinder Kaur, Quyen Anh Pham, and Lynn Epstein 2017. High quality DNA from Fusarium oxysporum conidia suitable for library preparation and long read sequencing with PacBio. protocols.io https://dx.doi.org/10.17504/protocols.io.i8ichue
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
Created: August 02, 2017
Last Modified: March 28, 2018
Protocol Integer ID: 7146
Abstract
This protocol is for isolation of high quality, high molecular weight DNA (20 kb and larger) that is suitable for PacBio library preparation. This protocol has been tested on lyophilized conidia from multiple isolates of Fusarium oxysporum, including f. sp. apii, f. sp. ciceris, and f. sp. lycopersici.
Guidelines
- When pipetting solutions with high molecular weight DNA, use low retention pipet tips with ca. 2 mm of the ends removed, i.e., wide-bore tips.
- Use low DNA-binding microfuge tubes.
Mixing is critical for purification, but avoid vigorous vortexing (which shears DNA)
We thank Martijn Rep, Benjamin Schwessinger, and Oanh Nguyen for advice. This protocol is modified from protocols from Dr. Martijn Rep, University of Amsterdam, and Dr. Benjamin Schwessinger, Australian National University.
Before start
Buffered Phenol Chloroform Isoamyl alcohol (P:C:I) ((25:24:1, saturated with 10 mM Tris, pH 8.0 and 1 mM EDTA) (Sigma P2069) [Note: use stocks that are less than 3 months old]
Sterilize all dry supplies by autoclaving.
Preparation of conidia for DNA extraction.
Preparation of conidia for DNA extraction.
Spread a Fusarium oxysporum conidial suspension onto 10 to 20, 100-mm-diam Petri dishes with potato dextrose agar.
Note
The goal is to obtain ca. 0.5 ml packed conidial volume, which yields ca. 250 mg lyophilized conidia.
Produce conidia by incubating dishes under fluorescent lights at ca. 25ºC for 6 to 7 days.
144:00:00 Incubation
To harvest the conidia, from each dish, pour 5 ml water on the culture, and gently rub the culture with a metal “hockey stick.” Repeat conidial collection with an additional 5 ml water.
Pour the dislodged conidia through two layers of Miracloth into 50 ml tubes.
Centrifuge the conidia at 4 °C at 3620 g for 15 min.
00:15:00 Centrifugation
Remove the supernatant (SN), resuspend and re-pellet.
Suspend the pellet in 3 to 5 ml water.
Transfer the conidial suspensions in 1 ml aliquots, each in a 2 ml microfuge tube.
Note
The goal is ca. 50 mg conidia dry wt/tube.
Centrifuge at 3620 g for 15 min.
00:15:00 Centrifugation
Remove water, freeze the conidial pellet at -70 ºC overnight (ON) and lyophilize it dry.
Note
Lyophilized conidia can be stored at -70 °C before grinding and extraction.
DNA extraction buffer for approx. 250 mg of lyophilized conidia.
DNA extraction buffer for approx. 250 mg of lyophilized conidia.
Use 1 ml of freshly prepared extraction buffer with RNase A per 50 mg of lyophilized Fusarium oxysporum conidia.
Prepare fresh just before extraction
For 5 ml extraction buffer add sequentially:
4 ml Milli-Q nuclease-free water
1 ml 5xRNB
240 mg PAS (Sodium 4-aminosalicylate dihydrate). Mix and then vortex until dissolved.
40 mg TIPS (Triisopropylnaphthalenesulfonic acid sodium) Mix and then vortex until dissolved.
Wait until the foam disappears. Add 5µl (50 µg) of RNase A (10mg/ml stock)
Conidial grinding.
Conidial grinding.
Cool a mortar (9 cm upper diam) with liquid N2.
Add additional liquid N2 to cool the sand.
Grind quickly with force for 15 to 20 sec. (1/3)
00:00:20 Grinding
Then add more liquid N2.
Grind again.
Add more liquid N2.
Grind again.
Transfer the ground conidia and sand mixture to a 2 ml microfuge tube (T#1) containing 1 ml of extraction buffer with RNase A from step 12.
Note
To optimize your grinding procedure, examine ground lyophilized conidia microscopically (Fig. 1); you need to grind sufficiently so that most of the conidia are cracked open. Any more grinding than that only shears the DNA.]
Note
We typically prepare 4 tubes, each with 50 mg lyophilized conidia per preparation. The tubes are pooled at the end of the preparation.
DNA extraction and purification.
DNA extraction and purification.
Label 3 additional tubes (T#2, T#3, & T#4) per 50 mg conidia.
Mix the T#1 tube from step 31 on a horizontal low speed (200 rpm) vortex mixer for 2 min.
00:02:00 Vortex
Incubate at room temperature (RT) for 30 min, with mixing by inverting tubes every 5 min.
00:30:00 Incubation at RT
00:05:00 Inverting
Add 6 µl (120 µg) of proteinase K to T#1.
Mix well on the low speed horizontal vortex mixer for 20 min at RT.
00:20:00 Vortex
Cool tubes on ice for 5 min.
00:05:00 Cooling on ice
Add an equal volume of ice-cold, buffered P:C:I (25:24:1)
Mix on the horizontal vortex mixer for 15 sec.
00:00:15 Vortex
Centrifuge at 15000g for 6 min at 4ºC.
00:06:00 Centrifugation
Note
The buffer is on top. Only use the lower PCI, here and in the subsequent step.
Transfer supernatant (SN) to a new 2 ml microfuge tube (T#2).
Add an equal volume of ice-cold, buffered P:C:I.
Mix on the horizontal vortex mixer for 2 min at 4ºC.
00:02:00 Vortex
Note
Always avoid transferring any of the interfaces.
Centrifuge at 15000g for 10 min at 4ºC.
00:10:00 Centrifugation
Transfer SN (Vol = 800 to 900µl) to a new 2 ml tube (T#3).
Add an equal volume of ice-cold chloroform:isoamyl alcohol (C:I) (24:1).
Mix on the horizontal vortex mixer for 15 sec at 4ºC.
00:00:15 Vortex
Centrifuge at 15000g for 5 min at 4°C.
00:05:00 Centrifugation
Very carefully, remove the SN without disturbing the interface to a new 2 ml tube (T#4).
Add 0.1 volume of ice-cold 4M NaCl to T#4 and mix by inverting the tube several times. DNA strings should be visible.
Add 1 volume of ice-cold isopropyl alcohol (IPA).
Mix well by inverting tubes gently at least 20 times.
Incubate on ice for 10 min.
00:10:00 Incubation on ice
Centrifuge at 10,000g for 30 min at 4ºC.
00:30:00 Centrifugation
Discard the SN. Wash the pellet with 1.5 ml ice-cold 70% ethanol. Centrifuge at 13000g for 5 min at RT.
Discard the SN. Wash the pellet with 1.5 ml ice-cold 70% ethanol. Centrifuge at 13000g for 5 min at RT.
Discard SN and centrifuge for 1 min at 13000g at RT. Remove residual ethanol with a fine Pippettor tip (e.g. P10 or P20).
Air dry the pellet for 15 to 20 min until there is no visible ethanol residue. Add 100 ul of EB (10mM Tris pH 8.5, Qiagen). Incubate at RT overnight. Next morning, incubate at 28 C on a platform orbit shaker at 150 rpm for 1 h.
Determine the DNA concentration with a Nanodrop (ND) and by Qubit. The Nanodrop 260/280 ratio should be 1.8-1.9 and the 260/230 ratio should be >2. Based on Qubit, the DNA quantity should be >200ng/ul. At this point, the concentrations of Qubit /ND is often ca. 0.1.
[Note: further cleanup is required until the Qubit/ND ratio >0.7.]
AMpure Bead Cleanup
AMpure Bead Cleanup
Perform an AMpure bead cleanup using either a 0.45X or 0.5X volume of beads as indicated below. Elute in 50 to100 ul EB.
Bring AMpure beads suspension to RT. Pipet out the beads slowly. Add 0.45X or 0.5X volume of the AMpure beads to the DNA.
Mix the beads in the DNA solution thoroughly by flicking the tube. Do not vortex.
Collect but do not pellet the beads at 7,000 g for 2 to 3 sec.
Allow the DNA to bind to the beads by shaking at 200 rpm on a horizontal vortex mixer for 10 min.
Incubate the tubes on a rotating wheel at 22 rpm for 20 min.
Centrifuge tubes at 7000 g for 10 sec to pellet the beads.
Place the tubes in a magnetic rack to hold the pellet on the side of the tube.
Slowly pipette off the SN. As a precautionary measure, save the SN in a new tube, but only as a backup–your DNA should be bound to the beads. Avoid disturbing the bead pellet.
With the beads in the magnetic rack, add 70% ethanol to the side of tube, i.e., avoid the pellet. Fill the 70% ethanol to the rim of the tube.10. Pipette off the ethanol and wash again with 70% ethanol as above. Remove all the ethanol without disturbing the bead pellet.
Remove tubes from the magnetic rack and centrifuge briefly at 7000 g for 30 sec to collect the residual ethanol. Place tubes on the magnetic rack and remove the ethanol with a fine tip Pipette (P20 or P10).
Check for any ethanol droplets. If present, repeat the previous step.
Remove the tubes from the magnetic rack. Open the caps. Add the amount of EB for >100ng/ul DNA (by Qubit). Mix the EB and beads gently by flicking until the suspension is homogenous. Centrifuge the tubes briefly at 7000 g for 30 sec. Place the tubes in the magnetic rack and let the DNA elute off the beads for 5 to 10 minutes. When all the brown beads are on the side with the magnet, carefully and slowly remove the gDNA into a clean 1.5 ml microfuge tube. Do not pipet out any of the magnetic beads.
Quantify DNA concentration and quality on Nanodrop and Qubit. For sufficient quality, with Nanodrop, the 260/280 ratio should be between 1.7 to 1.9 and the 260/230 ratio should be higher than 2.0. The Qubit/Nanodrop concentration ratio should be > 0.7X.
Based on Qubit, the DNA concentration should be >100ng/ul.
The size of the DNA should be checked by gel electrophoresis. We typically yield 80 ug DNA with mol wt. >48 kb per 250 mg lyophilized conidia.