Miniprep/Qiagen kit protocol

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Current revision (03:13, 20 November 2009) (view source)
 
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<html><h2>Solutions/reagents:</h2><ul type="circle"><li>bacterial culture</li><li> <a name="ice-cold Buffer P1"> ice-cold Buffer P1 <i><br><tab><div style="margin-right: 600px;">(50 mM Tris-HCl pH 8.0, 10 mM EDTA, 100 µg/ml RNaseA)</div></i></a></li><li> <a name="Buffer P2">Buffer P2 <i><br><tab><div style="margin-right: 600px;">(200 mM NaOH, 1% SDS)</div></i></a></li><li> <a name="Buffer N3">Buffer N3 <i><br><tab><div style="margin-right: 600px;">(4.2 M Gu-HCl, 0.9 M potassium acetate, pH 4.8)</div></i></a></li><li> <a name="Buffer PB">Buffer PB <i><br><tab><div style="margin-right: 600px;">(5 M Gu-HCl, 30% ethanol)</div></i></a></li><li> <a name="Buffer PE">Buffer PE <i><br><tab><div style="margin-right: 600px;">(10 mM Tris-HCl pH 7.5, 80% ethanol)</div></i></a></li><li> <a name="Buffer EB">Buffer EB <i><br><tab><div style="margin-right: 600px;">(10 mM Tris·Cl, pH 8.5)</div></i></a></li><li>QIAprep spin column</li></ul><h2>Equipment:</h2><ul type="circle"><li>Centrifuge</li><li>Microfuge</li><li>Flasks of appropriate volumes</li><li>Sterile 1.5-ml microcentrifuge tubes</li></ul><h2>Steps:</h2><ol><p><li>Measure out <b><font color=#357EC7>1.5 ml</font></b> of <font color=#357EC7>bacterial culture</font> into a sterile 1.5-ml microcentrifuge tube.<br>Centrifuge at <font color=#357EC7>maximum speed</font> for <b><font color=#357EC7>1 min</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>250 µl</font></b> of <a href="#ice-cold Buffer P1" ><font color=#357EC7>ice-cold Buffer P1</font></a>.<br>Resuspend the pellet by vortexing/by shaking vigorously.<br><font color = "#800517"><i>Ensure that RNase A has been added to Buffer P1. No cell clumps should be visible after resuspension of the pellet.</i></font><br></li></p><p><li>Add <b><font color=#357EC7>250 µl</font></b> of <a href="#Buffer P2" ><font color=#357EC7>Buffer P2</font></a>.<br>Close the tube tightly and invert the tube <b><font color=#357EC7>4 - 6 times</font></b> times.<br><font color = "#800517"><i>Mix gently by inverting the tube. Do not vortex, as this will result in shearing of genomic DNA. If necessary, continue inverting the tube until the solution becomes viscous and slightly clear.</i></font><br><font color=red>NOTE: Proceed to the next step within <b><font color=#357EC7>5 mins</font></b>!</font><br></li></p><p><li>Add <b><font color=#357EC7>250 µl</font></b> of <a href="#Buffer N3" ><font color=#357EC7>Buffer N3</font></a>.<br><font color=red>NOTE: Proceed to the next step within <b>immediately</b>!</font><br></li></p><p><li>Close the tube tightly and invert the tube <b><font color=#357EC7>4 - 6 times</font></b> times.<br><font color = "#800517"><i>To avoid localized precipitation, mix the solution gently but thoroughly, immediately after addition of Buffer N3. The solution should become cloudy.</i></font><br></li></p><p><li>Centrifuge at a speed of <font color=#357EC7>13000 rpm</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 the top layer.<br>Transfer top aqueous layer into QIAprep spin column.<br>Discard bottom layer.<br><font color = "#800517"><i>A compact white pellet will form the bottom layer.</i></font><br>Microfuge column for <b><font color=#357EC7>30 - 60 secs</font></b>.<br>Discard the flow-through.<br><font color = "#800517"><i>Centrifuging for 60 seconds produces good results.</i></font><br></li></p><p><b><font size=3>(Optional)</font></b><br>Add <b><font color=#357EC7>0.5 ml</font></b> of <a href="#Buffer PB" ><font color=#357EC7>Buffer PB</font></a> to column.<br>Microfuge column for <b><font color=#357EC7>30 - 60 secs</font></b>.<br>Discard the flow-through.<br><font color = "#800517"><i>This step is necessary to remove trace nuclease activity when using endA+ strains such as the JM series, HB101 and its derivatives, or any wild-type strain, which have high levels of nuclease activity or high carbohydrate content. Host strains such as XL-1 Blue and DH5?™ do not require this additional wash step. Although they call this step optional, it does not really hurt your yield and you may think you are working with an endA- strain when in reality you are not. Again for this step, spinning for 60 seconds produces good results.</i></font><br></li></p><p><li>Add <b><font color=#357EC7>0.75 ml</font></b> of <a href="#Buffer PE" ><font color=#357EC7>Buffer PE</font></a> to column.<br>Microfuge column for <b><font color=#357EC7>30 - 60 secs</font></b>.<br>Discard the flow-through.<br><font color = "#800517"><i>Centrifuging for 60 seconds produces good results.</i></font><br></li></p><p><li>Microfuge column for <b><font color=#357EC7>1 min</font></b>.<br>Discard the flow-through.<br><font color = "#800517"><i>This is to remove the residual wash bufer.</i></font><br><font color = "#800517"><i>IMPORTANT: Residual wash buffer will not be completely removed unless the flow-through is discarded before this additional centrifugation. Residual ethanol from Buffer PE may inhibit subsequent enzymatic reactions. They are right about this.</i></font><br></li></p><p><li>Transfer column into a clean 1.5 ml microcentrifuge tube.<br>Add <b><font color=#357EC7>50 µl</font></b> of <a href="#Buffer EB" ><font color=#357EC7>Buffer EB</font></a> to column.<br>Store at <b><font color=#357EC7><b><font color=#357EC7>room temperature</font></b></font></b> for <b><font color=#357EC7>1 min</font></b>.<br>Microfuge for <b><font color=#357EC7>1 min</font></b>.<br>Discard the column.<br><font color = "#800517"><i>The flow-through contains the DNA.</i></font><br><font color = "#800517"><i>If you are concerned about the concentration of the DNA, you can alternatively add 30 µl water to the center of the column, incubate at room temperature on the bench for 5 mins and then centrifuge for 1 min. This will increase the concentration of DNA in your final sample which can be useful in some cases. See notes below for why you should elute in water rather than the Buffer EB they recommend if you plan to sequence your sample. Even if you are not sequencing, it may be beneficial to elute in water. For instance, if you elute in buffer EB and you are using this DNA in a restriction digest, then the additional salts in your sample can affect the salt content of your digest. This may matter with some finicky enzymes.</i></font><br></li></p></ol></html>
+
<html><h2>Solutions/reagents:</h2><ul type="circle"><li>bacterial culture</li><li> <a name="ice-cold Buffer P1"> ice-cold Buffer P1 <i><br><tab><div style="margin-right: 600px;">(50 mM Tris-HCl pH 8.0, 10 mM EDTA, 100 µg/ml RNaseA)</div></i></a></li><li> <a name="Buffer P2">Buffer P2 <i><br><tab><div style="margin-right: 600px;">(200 mM NaOH, 1% SDS)</div></i></a></li><li> <a name="Buffer N3">Buffer N3 <i><br><tab><div style="margin-right: 600px;">(4.2 M Gu-HCl, 0.9 M potassium acetate, pH 4.8)</div></i></a></li><li> <a name="Buffer PB">Buffer PB <i><br><tab><div style="margin-right: 600px;">(5 M Gu-HCl, 30% ethanol)</div></i></a></li><li> <a name="Buffer PE">Buffer PE <i><br><tab><div style="margin-right: 600px;">(10 mM Tris-HCl pH 7.5, 80% ethanol)</div></i></a></li><li> <a name="Buffer EB">Buffer EB <i><br><tab><div style="margin-right: 600px;">(10 mM Tris·Cl, pH 8.5)</div></i></a></li><li>QIAprep spin column</li></ul><h2>Equipment:</h2><ul type="circle"><li>Centrifuge</li><li>Microfuge</li><li>Sterile 1.5-ml microcentrifuge tubes</li></ul><h2>Steps:</h2><ol><p><li>Measure out <b><font color=#357EC7>1.5 ml</font></b> of <font color=#357EC7>bacterial culture</font> into sterile 1.5-ml microcentrifuge tube (1).<br>Centrifuge at <font color=#357EC7>maximum speed</font> for <b><font color=#357EC7>1 min</font></b> at <b><font color=#357EC7>room temperature</font></b>, gently aspirate out the supernatant and discard it.<br>Measure out <b><font color=#357EC7>250 µl</font></b> of <a href="#ice-cold Buffer P1" ><font color=#357EC7>ice-cold Buffer P1</font></a> into pellet.<br>Resuspend pellet by vortexing/by shaking vigorously.<br><font color = "#800517"><i>Ensure that RNase A has been added to Buffer P1. No cell clumps should be visible after resuspension of the pellet.</i></font><br></li></p><p><li>Measure out <b><font color=#357EC7>250 µl</font></b> of <a href="#Buffer P2" ><font color=#357EC7>Buffer P2</font></a> into sterile 1.5-ml microcentrifuge tube (1).<br>Close the tube tightly and invert the tube <b><font color=#357EC7>4 - 6 times</font></b>.<br><font color = "#800517"><i>Mix gently by inverting the tube. Do not vortex, as this will result in shearing of genomic DNA. If necessary, continue inverting the tube until the solution becomes viscous and slightly clear.</i></font><br><font color=red>NOTE: Proceed to the next step within <b><font color=#357EC7>5 mins</font></b>!</font><br></li></p><p><li>Add <b><font color=#357EC7>250 µl</font></b> of <a href="#Buffer N3" ><font color=#357EC7>Buffer N3</font></a>.<br><font color=red>NOTE: Proceed to the next step within <b>immediately</b>!</font><br></li></p><p><li>Close the tube tightly and invert the tube <b><font color=#357EC7>4 - 6 times</font></b>.<br><font color = "#800517"><i>To avoid localized precipitation, mix the solution gently but thoroughly, immediately after addition of Buffer N3. The solution should become cloudy.</i></font><br></li></p><p><li>Centrifuge at a speed of <font color=#357EC7>13000 rpm</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 the top layer.<br>Transfer top aqueous layer into QIAprep spin column.<br>Discard bottom layer.<br><font color = "#800517"><i>A compact white pellet will form the bottom layer.</i></font><br>Microfuge column for <b><font color=#357EC7>30 - 60 secs</font></b>.<br>Discard the flow-through.<br><font color = "#800517"><i>Centrifuging for 60 seconds produces good results.</i></font><br></li></p><p><b><font size=3>(Optional)</font></b><br>Add <b><font color=#357EC7>0.5 ml</font></b> of <a href="#Buffer PB" ><font color=#357EC7>Buffer PB</font></a> to column.<br>Microfuge column for <b><font color=#357EC7>30 - 60 secs</font></b>.<br>Discard the flow-through.<br><font color = "#800517"><i>This step is necessary to remove trace nuclease activity when using endA+ strains such as the JM series, HB101 and its derivatives, or any wild-type strain, which have high levels of nuclease activity or high carbohydrate content. Host strains such as XL-1 Blue and DH5?™ do not require this additional wash step. Although they call this step optional, it does not really hurt your yield and you may think you are working with an endA- strain when in reality you are not. Again for this step, spinning for 60 seconds produces good results.</i></font><br></li></p><p><li>Add <b><font color=#357EC7>0.75 ml</font></b> of <a href="#Buffer PE" ><font color=#357EC7>Buffer PE</font></a> to column.<br>Microfuge column for <b><font color=#357EC7>30 - 60 secs</font></b>.<br>Discard the flow-through.<br><font color = "#800517"><i>Centrifuging for 60 seconds produces good results.</i></font><br></li></p><p><li>Microfuge column for <b><font color=#357EC7>1 min</font></b>.<br>Discard the flow-through.<br><font color = "#800517"><i>This is to remove the residual wash bufer.</i></font><br><font color = "#800517"><i>IMPORTANT: Residual wash buffer will not be completely removed unless the flow-through is discarded before this additional centrifugation. Residual ethanol from Buffer PE may inhibit subsequent enzymatic reactions. They are right about this.</i></font><br></li></p><p><li>Transfer column into sterile 1.5-ml microcentrifuge tube (2).<br>Add <b><font color=#357EC7>50 µl</font></b> of <a href="#Buffer EB" ><font color=#357EC7>Buffer EB</font></a> to column.<br>Store at <b><font color=#357EC7><b><font color=#357EC7>room temperature</font></b></font></b> for <b><font color=#357EC7>1 min</font></b>.<br>Microfuge for <b><font color=#357EC7>1 min</font></b>.<br>Discard the column.<br><font color = "#800517"><i>The flow-through contains the DNA.</i></font><br><font color = "#800517"><i>If you are concerned about the concentration of the DNA, you can alternatively add 30 µl water to the center of the column, incubate at room temperature on the bench for 5 mins and then centrifuge for 1 min. This will increase the concentration of DNA in your final sample which can be useful in some cases. See notes below for why you should elute in water rather than the Buffer EB they recommend if you plan to sequence your sample. Even if you are not sequencing, it may be beneficial to elute in water. For instance, if you elute in buffer EB and you are using this DNA in a restriction digest, then the additional salts in your sample can affect the salt content of your digest. This may matter with some finicky enzymes.</i></font><br></li></p></ol><p><b>TOTAL TIME REQUIRED FOR THE COMPLETION OF THE PROTOCOL :<font color=#357EC7>~ 22 mins</font></b></p></html>

Current revision

Solutions/reagents:

Equipment:

  • Centrifuge
  • Microfuge
  • Sterile 1.5-ml microcentrifuge tubes

Steps:

  1. Measure out 1.5 ml of bacterial culture into sterile 1.5-ml microcentrifuge tube (1).
    Centrifuge at maximum speed for 1 min at room temperature, gently aspirate out the supernatant and discard it.
    Measure out 250 µl of ice-cold Buffer P1 into pellet.
    Resuspend pellet by vortexing/by shaking vigorously.
    Ensure that RNase A has been added to Buffer P1. No cell clumps should be visible after resuspension of the pellet.
  2. Measure out 250 µl of Buffer P2 into sterile 1.5-ml microcentrifuge tube (1).
    Close the tube tightly and invert the tube 4 - 6 times.
    Mix gently by inverting the tube. Do not vortex, as this will result in shearing of genomic DNA. If necessary, continue inverting the tube until the solution becomes viscous and slightly clear.
    NOTE: Proceed to the next step within 5 mins!
  3. Add 250 µl of Buffer N3.
    NOTE: Proceed to the next step within immediately!
  4. Close the tube tightly and invert the tube 4 - 6 times.
    To avoid localized precipitation, mix the solution gently but thoroughly, immediately after addition of Buffer N3. The solution should become cloudy.
  5. Centrifuge at a speed of 13000 rpm for 10 mins at room temperature and aspirate out the top layer.
    Transfer top aqueous layer into QIAprep spin column.
    Discard bottom layer.
    A compact white pellet will form the bottom layer.
    Microfuge column for 30 - 60 secs.
    Discard the flow-through.
    Centrifuging for 60 seconds produces good results.
  6. (Optional)
    Add 0.5 ml of Buffer PB to column.
    Microfuge column for 30 - 60 secs.
    Discard the flow-through.
    This step is necessary to remove trace nuclease activity when using endA+ strains such as the JM series, HB101 and its derivatives, or any wild-type strain, which have high levels of nuclease activity or high carbohydrate content. Host strains such as XL-1 Blue and DH5?™ do not require this additional wash step. Although they call this step optional, it does not really hurt your yield and you may think you are working with an endA- strain when in reality you are not. Again for this step, spinning for 60 seconds produces good results.

  7. Add 0.75 ml of Buffer PE to column.
    Microfuge column for 30 - 60 secs.
    Discard the flow-through.
    Centrifuging for 60 seconds produces good results.
  8. Microfuge column for 1 min.
    Discard the flow-through.
    This is to remove the residual wash bufer.
    IMPORTANT: Residual wash buffer will not be completely removed unless the flow-through is discarded before this additional centrifugation. Residual ethanol from Buffer PE may inhibit subsequent enzymatic reactions. They are right about this.
  9. Transfer column into sterile 1.5-ml microcentrifuge tube (2).
    Add 50 µl of Buffer EB to column.
    Store at room temperature for 1 min.
    Microfuge for 1 min.
    Discard the column.
    The flow-through contains the DNA.
    If you are concerned about the concentration of the DNA, you can alternatively add 30 µl water to the center of the column, incubate at room temperature on the bench for 5 mins and then centrifuge for 1 min. This will increase the concentration of DNA in your final sample which can be useful in some cases. See notes below for why you should elute in water rather than the Buffer EB they recommend if you plan to sequence your sample. Even if you are not sequencing, it may be beneficial to elute in water. For instance, if you elute in buffer EB and you are using this DNA in a restriction digest, then the additional salts in your sample can affect the salt content of your digest. This may matter with some finicky enzymes.

TOTAL TIME REQUIRED FOR THE COMPLETION OF THE PROTOCOL :~ 22 mins

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