Line 913: | Line 913: | ||
<b>1.Production of cleared lysate</b> | <b>1.Production of cleared lysate</b> | ||
<br><br> | <br><br> | ||
− | < | + | <div style="text-indent:20px;"> |
− | 1. Isolation of the bacteria </ | + | 1. Isolation of the bacteria </div> |
− | < | + | <div style="text-indent:40px;"> |
1. harvest 1–5 ml (high-copy-number plasmid) or 10 ml (low-copy-number plasmid) | 1. harvest 1–5 ml (high-copy-number plasmid) or 10 ml (low-copy-number plasmid) | ||
of bacterial culture | of bacterial culture | ||
Line 926: | Line 926: | ||
<br> | <br> | ||
5. blot the inverted tube on a paper towel to remove excess media | 5. blot the inverted tube on a paper towel to remove excess media | ||
− | <br> </ | + | <br> </div> |
− | < | + | <div style="text-indent:20px;"> |
2. | 2. | ||
− | Resuspension of the cells </ | + | Resuspension of the cells </div> |
− | < | + | <div style="text-indent:40px;"> |
1. | 1. | ||
add 250 μl of Cell Resuspension Solution | add 250 μl of Cell Resuspension Solution | ||
Line 939: | Line 939: | ||
3. | 3. | ||
it is essential to thoroughly resuspend the cells | it is essential to thoroughly resuspend the cells | ||
− | <br></ | + | <br></div> |
− | < | + | <div style="text-indent:20px;"> |
3. | 3. | ||
− | Lysing </ | + | Lysing </div> |
− | < | + | <div style="text-indent:40px;"> |
1. | 1. | ||
add 250 μl of Cell Lysis Solution | add 250 μl of Cell Lysis Solution | ||
Line 952: | Line 952: | ||
3. | 3. | ||
incubate until the cell suspension clears (clear ≠ colorlessly) (approximately 1–5 minutes) | incubate until the cell suspension clears (clear ≠ colorlessly) (approximately 1–5 minutes) | ||
− | <br></ | + | <br></div> |
− | < | + | <div style="text-indent:20px;"> |
4. | 4. | ||
− | Splitting proteins </ | + | Splitting proteins </div> |
− | < | + | <div style="text-indent:40px;"> |
1. | 1. | ||
add 10 μl of Alkaline Protease Solution | add 10 μl of Alkaline Protease Solution | ||
Line 965: | Line 965: | ||
3. | 3. | ||
incubate for 5 minutes at room temperature | incubate for 5 minutes at room temperature | ||
− | <br></ | + | <br></div> |
− | < | + | <div style="text-indent:20px;"> |
5. | 5. | ||
− | Neutralization </ | + | Neutralization </div> |
− | < | + | <div style="text-indent:40px;"> |
1. | 1. | ||
add 350 μl of Neutralization Solution | add 350 μl of Neutralization Solution | ||
Line 975: | Line 975: | ||
2. | 2. | ||
immediately mix by inverting the tube 4 times - do not vortex | immediately mix by inverting the tube 4 times - do not vortex | ||
− | <br></ | + | <br></div> |
− | < | + | <div style="text-indent:20px;"> |
6. | 6. | ||
− | Isolation of the plasmids </ | + | Isolation of the plasmids </div> |
− | < | + | <div style="text-indent:40px;"> |
1. | 1. | ||
centrifuge the bacterial lysate at maximum speed (around 14,000 ×g) in a microcentrifuge for 10 minutes at room temperature </p> | centrifuge the bacterial lysate at maximum speed (around 14,000 ×g) in a microcentrifuge for 10 minutes at room temperature </p> | ||
− | <br> | + | <br></div> |
<b>2. Isolation of the plasmid DNA </b> | <b>2. Isolation of the plasmid DNA </b> | ||
<br> | <br> | ||
− | < | + | <div style="text-indent:20px;"> |
1. Transfer the cleared lysate (approximately 850 μl, Section 3.B, Step 6) to the | 1. Transfer the cleared lysate (approximately 850 μl, Section 3.B, Step 6) to the | ||
prepared Spin Column by decanting. Avoid disturbing or transferring any of the | prepared Spin Column by decanting. Avoid disturbing or transferring any of the | ||
− | white precipitate with the supernatant. </ | + | white precipitate with the supernatant. </div> |
<br> | <br> | ||
− | < | + | <div style="text-indent:40px;"> |
1. If the white precipitate is accidentally transferred to the Spin Column, pour | 1. If the white precipitate is accidentally transferred to the Spin Column, pour | ||
the Spin Column contents back into a sterile 1.5ml microcentrifuge tube | the Spin Column contents back into a sterile 1.5ml microcentrifuge tube | ||
and centrifuge for another 5–10 minutes at maximum speed. Transfer the | and centrifuge for another 5–10 minutes at maximum speed. Transfer the | ||
resulting supernatant into the same Spin Column that was used initially for | resulting supernatant into the same Spin Column that was used initially for | ||
− | this sample. The Spin Column can be reused but only for this sample.</ | + | this sample. The Spin Column can be reused but only for this sample.</div> |
<br> | <br> | ||
− | < | + | <div style="margin-left:20px;"> |
2. Centrifuge the supernatant at maximum speed in a microcentrifuge for 1 minute at | 2. Centrifuge the supernatant at maximum speed in a microcentrifuge for 1 minute at | ||
room temperature. Remove the Spin Column from the tube and discard the | room temperature. Remove the Spin Column from the tube and discard the | ||
flowthrough from the Collection Tube. Reinsert the Spin Column into the Collection | flowthrough from the Collection Tube. Reinsert the Spin Column into the Collection | ||
− | Tube. </ | + | Tube. </div> |
<br> | <br> | ||
− | < | + | <div style="text-indent:20px;"> |
− | 3. Wash the plasmid DNA | + | 3. Wash the plasmid DNA </div> |
<br> | <br> | ||
− | < | + | <div style="text-indent:40px;"> |
1. Add 750 μl of Column Wash Solution. | 1. Add 750 μl of Column Wash Solution. | ||
<br> | <br> | ||
Line 1,015: | Line 1,015: | ||
<br> | <br> | ||
4. Reinsert the Spin Column into the Collection Tube. | 4. Reinsert the Spin Column into the Collection Tube. | ||
− | <br></ | + | <br></div> |
− | < | + | <div style="text-indent:20px;"> |
− | 4. Wash again the plasmid DNA </ | + | 4. Wash again the plasmid DNA </div> |
<br> | <br> | ||
− | < | + | <div style="text-indent:40px;"> |
1. Add 250 μl of Column Wash Solution. | 1. Add 250 μl of Column Wash Solution. | ||
<br> | <br> | ||
Line 1,030: | Line 1,030: | ||
4. Transfer the Spin Column to a new, sterile 1.5ml microcentrifuge tube, being | 4. Transfer the Spin Column to a new, sterile 1.5ml microcentrifuge tube, being | ||
careful not to transfer any of the Column Wash Solution with the Spin Column. | careful not to transfer any of the Column Wash Solution with the Spin Column. | ||
− | <br></ | + | <br></div> |
− | < | + | <div style="text-indent:20px;"> |
− | 5. Elute the plasmid DNA </ | + | 5. Elute the plasmid DNA </div> |
<br> | <br> | ||
− | < | + | <div style="text-indent:40px;"> |
1. Add 50 μl of Nuclease-Free Water to the Spin Column, wait 5 minutes | 1. Add 50 μl of Nuclease-Free Water to the Spin Column, wait 5 minutes | ||
<br> | <br> | ||
2. Centrifuge at maximum speed for 1 minute at room temperature in a | 2. Centrifuge at maximum speed for 1 minute at room temperature in a | ||
microcentrifuge. | microcentrifuge. | ||
− | <br></ | + | <br></div> |
− | < | + | <div style="text-indent:20px;"> |
6. After eluting the DNA, remove the assembly from the 1.5ml microcentrifuge tube | 6. After eluting the DNA, remove the assembly from the 1.5ml microcentrifuge tube | ||
− | and discard the Spin Column.</ | + | and discard the Spin Column.</div> |
<br> | <br> | ||
Revision as of 20:00, 28 October 2017
Our research work
We are describing our research work. Below you can find the protocols we used.
Protocols