Filamentous fungi genomic DNA isolation protocol

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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>