Filamentous fungi genomic DNA isolation protocol

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(New page: <html><h2>Solutions/reagents:</h2><ul type="circle"><li> <a name="lysis buffer">lysis buffer <i><br><tab><div style="margin-right: 600px;">(50 mM Tris-HCL, 50 mM EDTA, 3% SDS, 1% 2-mercapt...)
Current revision (02:17, 20 November 2009) (view source)
 
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<html><h2>Solutions/reagents:</h2><ul type="circle"><li> <a name="lysis buffer">lysis buffer <i><br><tab><div style="margin-right: 600px;">(50 mM Tris-HCL, 50 mM EDTA, 3% SDS, 1% 2-mercaptoethanol (add just before use))</div></i></a></li><li>B-mercaptor</li><li> <a name="SEVAG">SEVAG <i><br><tab><div style="margin-right: 600px;">(chloroform:isoamyl alcohol, 24:1)</div></i></a></li><li> <a name="3M NaOAC">3M NaOAC <i><br><tab><div style="margin-right: 600px;">(pH 8.0)</div></i></a></li><li>isopropanol</li><li>EB</li><li>100 mg/ml RNAse</li><li>7.5 M ammonium acetate</li><li>100% EtOH</li><li>70% EtOH</li><li> <a name="TE buffer">TE buffer <i><br><tab><div style="margin-right: 600px;">(pH 8.0)</div></i></a></li><li> <a name="group lyophilized mycelium(wet)">group lyophilized mycelium(wet) <i><br><tab>(ground in liquid nitrogen)<br></i></a></li></ul><h2>Equipment:</h2><ul type="circle"><li>Incubator</li><li>Centrifuge</li><li>Sterile 1.5-ml microcentrifuge tubes</li><li>Eppendorf tubes</li></ul><h2>Steps:</h2><ol><p><li>Measure out <b><font color=#357EC7>1 g</font></b> of <a href="#group lyophilized mycelium(wet)" ><font color=#357EC7>group lyophilized mycelium(wet)</font></a> into a sterile 1.5-ml microcentrifuge tube.<br><font color = "#800517"><i>If using dry fungi, use 60- 100 mg. Alternatively you can grind 1 g of dried (vacuum filter mycelium first) in a mortar and pestle treating with liquid nitrogen 5-6 times. Pour the frozen powder into the eppendorf tube.</i></font><br><font color = "#800517"><i>Note: you only want to process about 1g in each eppendorf tube, if there is more than this, split to two separate tubes.</i></font><br></li></p><p><li>Add <b><font color=#357EC7>660 - 750 µl</font></b> of <a href="#lysis buffer" ><font color=#357EC7>lysis buffer</font></a>.<br>Add <b><font color=#357EC7>10 µl</font></b> of <font color=#357EC7>B-mercaptor</font>.<br>Vortex the mixture for a few secs.<br>Incubate at <b><font color=#357EC7>65°C</font></b> for <b><font color=#357EC7>1 hr</font></b>.<br><font color = "#800517"><i>Use a water bath for incubation.</i></font><br></li></p><p><li>Centrifuge at a speed of <font color=#357EC7>3400 rpm</font> for <b><font color=#357EC7>5 mins</font></b> at <b><font color=#357EC7><b><font color=#357EC7>room temperature</font></b></font></b> and aspirate out the top layer.<br>Transfer top aqueous layer into an Eppendorf tube.<br>Discard bottom layer.<br><font color = "#800517"><i>Do not take any of the cellular debris from the interface. Don't be greedy this prep extracts a lot of DNA.</i></font><br></li></p><p><li>Add <b><font color=#357EC7>700 µl</font></b> of <a href="#SEVAG" ><font color=#357EC7>SEVAG</font></a>.<br><font color = "#800517"><i>Adjust volume if needed to meet a 1:1 ratio of SEVAG and aqueous phase.</i></font><br>Vortex the mixture for a few secs.<br><font color = "#800517"><i>Note: some people top off with EB buffer so that total volume is so that volume is equal among the samples for spinning. Be careful as sometimes tops are loosened by chloroform.</i></font><br>Centrifuge at a speed of <font color=#357EC7>12000 Xg</font> for <b><font color=#357EC7>10 mins</font></b> at <b><font color=#357EC7><b><font color=#357EC7>room temperature</font></b></font></b> and aspirate out <b><font color=#357EC7>550 - 600 µl</font></b> of top layer.<br>Transfer top aqueous layer into an Eppendorf tube.<br>Discard bottom layer.<br></li></p><p><li><font color = "#800517"><i>Note: these last two steps (SEVAG, spin, transfer) can be repeated to insure cleaner DNA prep depending on your needs and pipette techniques.</i></font><br></li></p><p><li>Add <b><font color=#357EC7>20 µl</font></b> of <a href="#3M NaOAC" ><font color=#357EC7>3M NaOAC</font></a>.<br>Add <font color=#357EC7>isopropanol</font> to top aqueous layer.<br><font color = "#800517"><i>Top off with isopropanol.</i></font><br>Close the tube tightly and gently mix the contents by inverting the tube.<br><font color = "#800517"><i>You should see DNA "ropes" precipitating.</i></font><br></li></p><p><li>Centrifuge at <font color=#357EC7>maximum speed</font> for <b><font color=#357EC7>2 mins</font></b> at <b><font color=#357EC7>room temperature</font></b>, gently aspirate out the supernatant and discard it.<br>Stand the tube containing pellet for <b><font color=#357EC7>1 min</font></b> in an inverted position on a paper towel to allow all of the fluid to drain away.<br></li></p><p><li>Add <b><font color=#357EC7>300 µl</font></b> of <font color=#357EC7>EB</font>.<br>Add <b><font color=#357EC7>1 µl</font></b> of <font color=#357EC7>100 mg/ml RNAse</font>.<br>Store at <b><font color=#357EC7>65°C</font></b> for <b><font color=#357EC7>10 - 15 mins</font></b>.<br>Vortex the mixture for a few secs.<br><font color = "#800517"><i>Finger vortex the tube to prevent shearing of DNA.</i></font><br></li></p><p><b><font size=3>(Optional)</font></b><br><font color = "red"><i>Further cleanup with the PEG DNA protocol.</i></font><br></li></p><p><li>Add <b><font color=#357EC7>250 µl</font></b> of <font color=#357EC7>7.5 M ammonium acetate</font>.<br>Centrifuge at <font color=#357EC7>maximum speed</font> for <b><font color=#357EC7>5 mins</font></b> at <b><font color=#357EC7><b><font color=#357EC7>room temperature</font></b></font></b> and aspirate out the top layer.<br>Transfer top aqueous layer into an Eppendorf tube.<br>Discard bottom layer.<br><font color = "#800517"><i>This pellets the protein debris.</i></font><br>Add <b><font color=#357EC7>750 µl</font></b> of <font color=#357EC7>isopropanol</font>.<br></li></p><p><li>Centrifuge at <font color=#357EC7>maximum speed</font> for <b><font color=#357EC7>30 - 120 secs</font></b> at <b><font color=#357EC7>room temperature</font></b>, gently aspirate out the supernatant and discard it.<br>Add <b><font color=#357EC7>1 ml</font></b> of <font color=#357EC7>100% EtOH</font>.<br>Vortex the mixture for a few secs.<br>Centrifuge at <font color=#357EC7>maximum speed</font> for <b><font color=#357EC7>30 - 120 secs</font></b> at <b><font color=#357EC7>room temperature</font></b>, gently aspirate out the supernatant and discard it.<br>Add <b><font color=#357EC7>1 ml</font></b> of <font color=#357EC7>70% EtOH</font>.<br>Vortex the mixture for a few secs.<br>Centrifuge at <font color=#357EC7>maximum speed</font> for <b><font color=#357EC7>30 - 120 secs</font></b> at <b><font color=#357EC7>room temperature</font></b>, gently aspirate out the supernatant and discard it.<br></li></p><p><li><p>Option 1: Dry the pellet in vacuum oven at 50°C for at most <b><font color=#357EC7>15 mins</font></b>.<br>(or)<br>Option 2: Dry the pellet in speed vac. <br></p><p></li></p><p><li>Add <font color=#357EC7>TE buffer</font> to pellet.<br>Resuspend the pellet by vortexing/by shaking vigorously.<br></li></p></ol></html>
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<html><h2>Solutions/reagents:</h2><ul type="circle"><li> <a name="lysis buffer">lysis buffer <i><br><tab><div style="margin-right: 600px;">(50 mM Tris-HCL, 50 mM EDTA, 3% SDS, 1% 2-mercaptoethanol (add just before use))</div></i></a></li><li>B-mercaptor</li><li> <a name="SEVAG">SEVAG <i><br><tab><div style="margin-right: 600px;">(chloroform:isoamyl alcohol, 24:1)</div></i></a></li><li> <a name="3M NaOAC">3M NaOAC <i><br><tab><div style="margin-right: 600px;">(pH 8.0)</div></i></a></li><li>isopropanol</li><li>EB</li><li>100 mg/ml RNAse</li><li>7.5 M ammonium acetate</li><li>100% EtOH</li><li>70% EtOH</li><li> <a name="TE buffer">TE buffer <i><br><tab><div style="margin-right: 600px;">(pH 8.0)</div></i></a></li><li> <a name="group lyophilized mycelium(wet)">group lyophilized mycelium(wet) <i><br><tab>(ground in liquid nitrogen)<br></i></a></li></ul><h2>Equipment:</h2><ul type="circle"><li>Incubator</li><li>Centrifuge</li><li>Sterile 1.5-ml microcentrifuge tubes</li><li>Eppendorf tubes</li></ul><h2>Steps:</h2><ol><p><li>Measure out <b><font color=#357EC7>1 g</font></b> of <a href="#group lyophilized mycelium(wet)" ><font color=#357EC7>group lyophilized mycelium(wet)</font></a> into sterile 1.5-ml microcentrifuge tube (1).<br><font color = "#800517"><i>If using dry fungi, use 60- 100 mg. Alternatively you can grind 1 g of dried (vacuum filter mycelium first) in a mortar and pestle treating with liquid nitrogen 5-6 times. Pour the frozen powder into the eppendorf tube.</i></font><br><font color = "#800517"><i>Note: you only want to process about 1g in each eppendorf tube, if there is more than this, split to two separate tubes.</i></font><br></li></p><p><li>Add <b><font color=#357EC7>660 - 750 µl</font></b> of <a href="#lysis buffer" ><font color=#357EC7>lysis buffer</font></a>.<br>Add <b><font color=#357EC7>10 µl</font></b> of <font color=#357EC7>B-mercaptor</font>.<br>Vortex the mixture for a few secs.<br>Incubate at <b><font color=#357EC7>65°C</font></b> for <b><font color=#357EC7>1 hr</font></b>.<br><font color = "#800517"><i>Use a water bath for incubation.</i></font><br></li></p><p><li>Centrifuge at a speed of <font color=#357EC7>3400 rpm</font> for <b><font color=#357EC7>5 mins</font></b> at <b><font color=#357EC7><b><font color=#357EC7>room temperature</font></b></font></b> and aspirate out the top layer.<br>Transfer top aqueous layer into Eppendorf tube (1).<br>Discard bottom layer.<br><font color = "#800517"><i>Do not take any of the cellular debris from the interface. Don't be greedy this prep extracts a lot of DNA.</i></font><br></li></p><p><li>Measure out <b><font color=#357EC7>700 µl</font></b> of <a href="#SEVAG" ><font color=#357EC7>SEVAG</font></a> into Eppendorf tube (1).<br><font color = "#800517"><i>Adjust volume if needed to meet a 1:1 ratio of SEVAG and aqueous phase.</i></font><br>Vortex the mixture for a few secs.<br><font color = "#800517"><i>Note: some people top off with EB buffer so that total volume is so that volume is equal among the samples for spinning. Be careful as sometimes tops are loosened by chloroform.</i></font><br>Centrifuge at a speed of <font color=#357EC7>12000 Xg</font> for <b><font color=#357EC7>10 mins</font></b> at <b><font color=#357EC7><b><font color=#357EC7>room temperature</font></b></font></b> and aspirate out <b><font color=#357EC7>550 - 600 µl</font></b> of top layer.<br>Transfer top aqueous layer into Eppendorf tube (2).<br>Discard bottom layer.<br></li></p><p><li><font color = "#800517"><i>Note: these last two steps (SEVAG, spin, transfer) can be repeated to insure cleaner DNA prep depending on your needs and pipette techniques.</i></font><br></li></p><p><li>Measure out <b><font color=#357EC7>20 µl</font></b> of <a href="#3M NaOAC" ><font color=#357EC7>3M NaOAC</font></a> into Eppendorf tube (2).<br>Add <font color=#357EC7>isopropanol</font> to 3M NaOAC.<br><font color = "#800517"><i>Top off with isopropanol.</i></font><br>Close the tube tightly and gently mix the contents by inverting the tube.<br><font color = "#800517"><i>You should see DNA "ropes" precipitating.</i></font><br></li></p><p><li>Centrifuge at <font color=#357EC7>maximum speed</font> for <b><font color=#357EC7>2 mins</font></b> at <b><font color=#357EC7>room temperature</font></b>, gently aspirate out the supernatant and discard it.<br>Stand the tube containing pellet for <b><font color=#357EC7>1 min</font></b> in an inverted position on a paper towel to allow all of the fluid to drain away.<br></li></p><p><li>Add <b><font color=#357EC7>300 µl</font></b> of <font color=#357EC7>EB</font>.<br>Add <b><font color=#357EC7>1 µl</font></b> of <font color=#357EC7>100 mg/ml RNAse</font>.<br>Store at <b><font color=#357EC7>65°C</font></b> for <b><font color=#357EC7>10 - 15 mins</font></b>.<br>Vortex the mixture for a few secs.<br><font color = "#800517"><i>Finger vortex the tube to prevent shearing of DNA.</i></font><br></li></p><p><b><font size=3>(Optional)</font></b><br><font color = "red"><i>Further cleanup with the PEG DNA protocol.</i></font><br></li></p><p><li>Add <b><font color=#357EC7>250 µl</font></b> of <font color=#357EC7>7.5 M ammonium acetate</font>.<br>Centrifuge at <font color=#357EC7>maximum speed</font> for <b><font color=#357EC7>5 mins</font></b> at <b><font color=#357EC7><b><font color=#357EC7>room temperature</font></b></font></b> and aspirate out the top layer.<br>Transfer top aqueous layer into Eppendorf tube (3).<br>Discard bottom layer.<br><font color = "#800517"><i>This pellets the protein debris.</i></font><br>Measure out <b><font color=#357EC7>750 µl</font></b> of <font color=#357EC7>isopropanol</font> into Eppendorf tube (3).<br></li></p><p><li>Centrifuge at <font color=#357EC7>maximum speed</font> for <b><font color=#357EC7>30 - 120 secs</font></b> at <b><font color=#357EC7>room temperature</font></b>, gently aspirate out the supernatant and discard it.<br>Add <b><font color=#357EC7>1 ml</font></b> of <font color=#357EC7>100% EtOH</font>.<br>Vortex the mixture for a few secs.<br>Centrifuge at <font color=#357EC7>maximum speed</font> for <b><font color=#357EC7>30 - 120 secs</font></b> at <b><font color=#357EC7>room temperature</font></b>, gently aspirate out the supernatant and discard it.<br>Add <b><font color=#357EC7>1 ml</font></b> of <font color=#357EC7>70% EtOH</font>.<br>Vortex the mixture for a few secs.<br>Centrifuge at <font color=#357EC7>maximum speed</font> for <b><font color=#357EC7>30 - 120 secs</font></b> at <b><font color=#357EC7>room temperature</font></b>, gently aspirate out the supernatant and discard it.<br></li></p><p><li><p><b>Option 1: </b>Dry the pellet in vacuum oven at 50°C for at most <b><font color=#357EC7>15 mins</font></b>.<br>(or)<br><b>Option 2: </b>Dry the pellet in speed vac. <br></p><p></li></p><p><li>Add <font color=#357EC7>TE buffer</font> to pellet.<br>Resuspend pellet by vortexing/by shaking vigorously.<br></li></p></ol><p><b>TOTAL TIME REQUIRED FOR THE COMPLETION OF THE PROTOCOL :<font color=#357EC7>~ 1 hr, 59 mins</font></b></p></html>

Current revision

Solutions/reagents:

Equipment:

  • Incubator
  • Centrifuge
  • Sterile 1.5-ml microcentrifuge tubes
  • Eppendorf tubes

Steps:

  1. Measure out 1 g of group lyophilized mycelium(wet) into sterile 1.5-ml microcentrifuge tube (1).
    If using dry fungi, use 60- 100 mg. Alternatively you can grind 1 g of dried (vacuum filter mycelium first) in a mortar and pestle treating with liquid nitrogen 5-6 times. Pour the frozen powder into the eppendorf tube.
    Note: you only want to process about 1g in each eppendorf tube, if there is more than this, split to two separate tubes.
  2. Add 660 - 750 µl of lysis buffer.
    Add 10 µl of B-mercaptor.
    Vortex the mixture for a few secs.
    Incubate at 65°C for 1 hr.
    Use a water bath for incubation.
  3. Centrifuge at a speed of 3400 rpm for 5 mins at room temperature and aspirate out the top layer.
    Transfer top aqueous layer into Eppendorf tube (1).
    Discard bottom layer.
    Do not take any of the cellular debris from the interface. Don't be greedy this prep extracts a lot of DNA.
  4. Measure out 700 µl of SEVAG into Eppendorf tube (1).
    Adjust volume if needed to meet a 1:1 ratio of SEVAG and aqueous phase.
    Vortex the mixture for a few secs.
    Note: some people top off with EB buffer so that total volume is so that volume is equal among the samples for spinning. Be careful as sometimes tops are loosened by chloroform.
    Centrifuge at a speed of 12000 Xg for 10 mins at room temperature and aspirate out 550 - 600 µl of top layer.
    Transfer top aqueous layer into Eppendorf tube (2).
    Discard bottom layer.
  5. Note: these last two steps (SEVAG, spin, transfer) can be repeated to insure cleaner DNA prep depending on your needs and pipette techniques.
  6. Measure out 20 µl of 3M NaOAC into Eppendorf tube (2).
    Add isopropanol to 3M NaOAC.
    Top off with isopropanol.
    Close the tube tightly and gently mix the contents by inverting the tube.
    You should see DNA "ropes" precipitating.
  7. Centrifuge at maximum speed for 2 mins at room temperature, gently aspirate out the supernatant and discard it.
    Stand the tube containing pellet for 1 min in an inverted position on a paper towel to allow all of the fluid to drain away.
  8. Add 300 µl of EB.
    Add 1 µl of 100 mg/ml RNAse.
    Store at 65°C for 10 - 15 mins.
    Vortex the mixture for a few secs.
    Finger vortex the tube to prevent shearing of DNA.
  9. (Optional)
    Further cleanup with the PEG DNA protocol.

  10. Add 250 µl of 7.5 M ammonium acetate.
    Centrifuge at maximum speed for 5 mins at room temperature and aspirate out the top layer.
    Transfer top aqueous layer into Eppendorf tube (3).
    Discard bottom layer.
    This pellets the protein debris.
    Measure out 750 µl of isopropanol into Eppendorf tube (3).
  11. Centrifuge at maximum speed for 30 - 120 secs at room temperature, gently aspirate out the supernatant and discard it.
    Add 1 ml of 100% EtOH.
    Vortex the mixture for a few secs.
    Centrifuge at maximum speed for 30 - 120 secs at room temperature, gently aspirate out the supernatant and discard it.
    Add 1 ml of 70% EtOH.
    Vortex the mixture for a few secs.
    Centrifuge at maximum speed for 30 - 120 secs at room temperature, gently aspirate out the supernatant and discard it.
  12. Option 1: Dry the pellet in vacuum oven at 50°C for at most 15 mins.
    (or)
    Option 2: Dry the pellet in speed vac.

  13. Add TE buffer to pellet.
    Resuspend pellet by vortexing/by shaking vigorously.

TOTAL TIME REQUIRED FOR THE COMPLETION OF THE PROTOCOL :~ 1 hr, 59 mins

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