Miniprep - GET Buffer protocol: Difference between revisions
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<html><h1 style="font-size = 25px;">Miniprep - GET Buffer</h1><h2 style="margin-top:50px;">Solutions/reagents:</h2><ul type="circle"><li>overnight culture</li><li> <a name="ice-cold GET buffer"> ice-cold GET buffer <i><br><tab><div style="margin-right: 600px;">(50 mM glucose (MW 180), 10mM EDTA, 25 mM Tris-HCl pH 8)</div></i></a></li><li>0.2M NaOH stored at room temperature</li><li> <a name="ice-cold potassium acetate solution"> ice-cold potassium acetate solution <i><br><tab><div style="margin-right: 600px;">(3 M potassium acetate, 1.8 M acetic acid, no pH adjustment)</div></i></a></li><li>95% / 100% ethanol</li><li>70% EtOH</li><li>TE buffer</li><li>distilled water</li><li> <a name="PCA solution">PCA solution <i><br><tab><div style="margin-right: 600px;">((optional)50 parts phenol, 49 parts chloroform, and 1 part amyl-alcohol)</div></i></a></li><li>SDS</li><li> <a name="lysozyme">lysozyme <i><br><tab>(optional)<br></i></a></li></ul><h2>Equipment:</h2><ul type="circle"><li>Centrifuge</li><li>Sterile 2-ml microcentrifuge tubes</li></ul><h2>Steps:</h2><ol><p><li>Measure out <b><font color=#357EC7>2 ml</font></b> of <font color=#357EC7>overnight culture</font> into a sterile 2-ml microcentrifuge tube.<br></li></p><p><li>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></li></p><p><li>Add <b><font color=#357EC7>100 µl</font></b> of <a href="#ice-cold GET buffer" ><font color=#357EC7>ice-cold GET buffer</font></a>.<br>Resuspend the pellet by vortexing/by shaking vigorously.<br></li></p><p><b><font size=3>(Optional)</font></b><br>Add <b><font color=#357EC7> 10 mg</font></b> of <a href="#lysozyme" ><font color=#357EC7>lysozyme</font></a>.<br>Store at <b><font color=#357EC7><b><font color=#357EC7>room temperature</font></b></font></b> for <b><font color=#357EC7>30 mins</font></b>.<br><font color = "#800517"><i>This step is essential for lysing gram-positive cells.</i></font><br></li></p><p><li>Measure out <b><font color=#357EC7>200 µl</font></b> of <font color=#357EC7>0.2M NaOH</font> into a sterile 2-ml microcentrifuge tube.<br>Add <b><font color=#357EC7> 2 mg</font></b> of <font color=#357EC7>SDS</font>.<br>Vortex the mixture for a few secs.<br></li></p><p><li>Add <font color=#357EC7>alkaline SDS solution</font> to cell suspension.<br>Close the tube tightly and gently mix the contents by inverting the tube.<br><font color = "#800517"><i>DO NOT VORTEX! The solution should become clear.</i></font><br></li></p><p><li>Add <b><font color=#357EC7>150 µl</font></b> of <a href="#ice-cold potassium acetate solution" ><font color=#357EC7>ice-cold potassium acetate solution</font></a>.<br>Close the tube tightly and gently mix the contents by inverting the tube.<br><font color = "#800517"><i>DO NOT VORTEX! A precipitate should form.</i></font><br></li></p><p><li>Store the tube <b><font color=#357EC7>on ice</font></b> for <b><font color=#357EC7>3 - 5 mins</font></b>.<br></li></p><p><li>Centrifuge at <font color=#357EC7>maximum speed</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>400 µl</font></b> of top layer.<br>Transfer top aqueous layer into a sterile 2-ml microcentrifuge tube.<br>Discard bottom layer.<br><font color = "#800517"><i>DO NOT PICK UP ANY PRECIPITATE!!!</i></font><br></li></p><p><b><font size=3>(Optional)</font></b><br>Add <b><font color=#357EC7>400 µl</font></b> of <a href="#PCA solution" ><font color=#357EC7>PCA solution</font></a>.<br>Close the tube tightly and gently mix the contents by inverting the tube.<br>Centrifuge at <font color=#357EC7>maximum speed</font> for <b><font color=#357EC7>3 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 a sterile 2-ml microcentrifuge tube.<br>Discard bottom layer.<br><font color = "#800517"><i>This helps remove any residual proteins.</i></font><br></li></p><p><li>Add <b><font color=#357EC7>900 µl</font></b> of <font color=#357EC7>95% / 100% ethanol</font>.<br><font color = "#800517"><i>This is to precipitate the plasmid DNA.</i></font><br></li></p><p><li>Store at <b><font color=#357EC7>-80°C</font></b> for <b><font color=#357EC7>30 mins</font></b>.<br><font color = "#800517"><i>Use the -80°C freezer.</i></font><br></li></p><p><li>Centrifuge at <font color=#357EC7>maximum speed</font> for <b><font color=#357EC7>10 mins</font></b> at <b><font color=#357EC7>room temperature</font></b>, gently aspirate out the supernatant and discard it.<br></li></p><p><li>Add <b><font color=#357EC7>1 ml</font></b> of <font color=#357EC7>70% EtOH</font>.<br>Store at <b><font color=#357EC7><b><font color=#357EC7>room temperature</font></b></font></b> for <b><font color=#357EC7>3 mins</font></b>.<br></li></p><p><li>Centrifuge at <font color=#357EC7>maximum speed</font> for <b><font color=#357EC7>3 mins</font></b> at <b><font color=#357EC7>room temperature</font></b>, gently aspirate out the supernatant and discard it.<br><font color = "#800517"><i>Make sure the pellet is toward the outside.</i></font><br></li></p><p><li>Dry the pellet in air for <b><font color=#357EC7>10 - 15 mins</font></b>.<br></li></p><p><li><font color = "#800517"><i>Make sure the pellet is completely dry before this step.</i></font><br><p>Option 1: Add <b><font color=#357EC7>20 µl</font></b> of <font color=#357EC7>TE buffer</font>.<br>(or)<br>Option 2: Add <b><font color=#357EC7>20 µl</font></b> of <font color=#357EC7>distilled water</font>.<br></p><p>Resuspend the pellet by vortexing/by shaking vigorously.<br><font color = "#800517"><i> The DNA will contain RNA contamination, which can be removed by resuspending in TE with RNAse.</i></font><br></li></p></ol></html> | |||
Revision as of 12:18, 22 September 2009
<html>
Miniprep - GET Buffer
Solutions/reagents:
- overnight culture
- <a name="ice-cold GET buffer"> ice-cold GET buffer
<tab>(50 mM glucose (MW 180), 10mM EDTA, 25 mM Tris-HCl pH 8)</a> - 0.2M NaOH stored at room temperature
- <a name="ice-cold potassium acetate solution"> ice-cold potassium acetate solution
<tab>(3 M potassium acetate, 1.8 M acetic acid, no pH adjustment)</a> - 95% / 100% ethanol
- 70% EtOH
- TE buffer
- distilled water
- <a name="PCA solution">PCA solution
<tab>((optional)50 parts phenol, 49 parts chloroform, and 1 part amyl-alcohol)</a> - SDS
- <a name="lysozyme">lysozyme
<tab>(optional)
</a>
Equipment:
- Centrifuge
- Sterile 2-ml microcentrifuge tubes
Steps:
- Measure out 2 ml of overnight culture into a sterile 2-ml microcentrifuge tube.
- Centrifuge at maximum speed for 1 min at room temperature, gently aspirate out the supernatant and discard it.
- Add 100 µl of <a href="#ice-cold GET buffer" >ice-cold GET buffer</a>.
Resuspend the pellet by vortexing/by shaking vigorously. - Measure out 200 µl of 0.2M NaOH into a sterile 2-ml microcentrifuge tube.
Add 2 mg of SDS.
Vortex the mixture for a few secs. - Add alkaline SDS solution to cell suspension.
Close the tube tightly and gently mix the contents by inverting the tube.
DO NOT VORTEX! The solution should become clear. - Add 150 µl of <a href="#ice-cold potassium acetate solution" >ice-cold potassium acetate solution</a>.
Close the tube tightly and gently mix the contents by inverting the tube.
DO NOT VORTEX! A precipitate should form. - Store the tube on ice for 3 - 5 mins.
- Centrifuge at maximum speed for 10 mins at room temperature and aspirate out 400 µl of top layer.
Transfer top aqueous layer into a sterile 2-ml microcentrifuge tube.
Discard bottom layer.
DO NOT PICK UP ANY PRECIPITATE!!! - Add 900 µl of 95% / 100% ethanol.
This is to precipitate the plasmid DNA. - Store at -80°C for 30 mins.
Use the -80°C freezer. - Centrifuge at maximum speed for 10 mins at room temperature, gently aspirate out the supernatant and discard it.
- Add 1 ml of 70% EtOH.
Store at room temperature for 3 mins. - Centrifuge at maximum speed for 3 mins at room temperature, gently aspirate out the supernatant and discard it.
Make sure the pellet is toward the outside. - Dry the pellet in air for 10 - 15 mins.
- Make sure the pellet is completely dry before this step.
Option 1: Add 20 µl of TE buffer.
(or)
Option 2: Add 20 µl of distilled water.Resuspend the pellet by vortexing/by shaking vigorously.
The DNA will contain RNA contamination, which can be removed by resuspending in TE with RNAse.
(Optional)
Add 10 mg of <a href="#lysozyme" >lysozyme</a>.
Store at room temperature for 30 mins.
This step is essential for lysing gram-positive cells.
(Optional)
Add 400 µl of <a href="#PCA solution" >PCA solution</a>.
Close the tube tightly and gently mix the contents by inverting the tube.
Centrifuge at maximum speed for 3 mins at room temperature and aspirate out the top layer.
Transfer top aqueous layer into a sterile 2-ml microcentrifuge tube.
Discard bottom layer.
This helps remove any residual proteins.
</html>
