Difference between revisions of "Team:Bulgaria/PROTOCOLS"

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<h1>PureLink® Quick Gel Extraction and PCR Purification Combo Kit</h1>
 +
<p><strong><i>Purifying PCR fragments or reaction clean-up</i></strong>
 +
<br><br>
 +
<strong>1. </strong>Combine. Add 4 volumes of Binding Buffer (B2) with isopropanol to 1 volume of a PCR sample (50–100 uL). Mix well.<br>
 +
<strong>2. </strong>Load. Pipet the sample into a PureLink® Clean-up Spin Column in a Wash Tube. Centrifuge the column at >10,000 × g for 1 minute. Discard the flow-through.<br>
 +
<strong>3. </strong>Wash. Re-insert the column into the Wash Tube and add 650 uL Wash Buffer (W1) with ethanol. Centrifuge the column at >10,000 × g for 1 minute.<br>
 +
<strong>4. </strong>Remove ethanol. Discard the flow-through and place the column in the same Wash Tube. Centrifuge the column at maximum speed for 2–3 minutes.<br>
 +
<strong>5. </strong>Elute. Place the column into a clean 1.5-mL Elution Tube. Add 50 uL Elution Buffer (E1) to the column. Incubate the column at room temperature for 1 minute. Centrifuge the column at maximum speed for 1 minute.<br>
 +
<strong>6. </strong>Store. The elution tube contains the purified PCR product. Store the purified DNA at 4°C for immediate use or at -20°C for long-term storage.
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</p>
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<div class="contentHolder" id="PROJECTPROTOCOLS">
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<h1>Miniprep Plasmid Purification</h1>
 +
<p>
 +
<strong>1. </strong>Resuspend the pelleted cells in 250 uL of the Resuspension Solution. Transfer the cell suspension to a microcentrifuge tube. The bacteria should be resuspended completely by vortexing or pipetting up and down until no cell clumps remain.<br>
 +
<strong>2. </strong>Add 250 uL of the Lysis Solution and mix thoroughly by inverting the tube 4-6 times until the solution becomes viscous and slightly clear. Do not vortex to avoid shearing of chromosomal DNA. Do not incubate for more than 5 min to avoid denaturation of supercoiled plasmid DNA.<br>
 +
<strong>3. </strong>Add 350 uL of the Neutralization Solution and mix immediately and thoroughly by inverting the tube 4-6 times. The neutralized bacterial lysate should become cloudy.<br>
 +
<strong>4. </strong>Centrifuge for 5 min to pellet cell debris and chromosomal DNA.<br>
 +
<strong>5. </strong>Transfer the supernatant to the supplied GeneJET spin column by decanting or pipetting. Avoid disturbing or transferring the white precipitate.<br>
 +
<strong>6. </strong>Centrifuge for 1 min. Discard the flow-through and place the column back into the same collection tube.<br>
 +
<strong>7. </strong>Add 500 uL of the Wash Solution to the GeneJET spin column. Centrifuge for 30-60 seconds and discard the flow-through. Place the column back into the same collection tube.<br>
 +
<strong>8. </strong>Repeat the wash procedure using 500 uL of the Wash Solution.<br>
 +
<strong>9. </strong>Discard the flow-through and centrifuge for an additional 1 min to remove residual Wash Solution. This step is essential to avoid residual ethanol in plasmid preps.<br>
 +
<strong>10. </strong>Transfer the GeneJET spin column into a fresh 1.5 mL microcentrifuge tube (not included). Add 50 uL of the Elution Buffer to the center of GeneJET spin column membrane to elute the plasmid DNA. Take care not to contact the membrane with the pipette tip. Incubate for 2 min at room temperature and centrifuge for 2 min.. For elution of plasmids or cosmids >20 kb, prewarm Elution Buffer to 70°C before applying to silica membrane.<br>
 +
<strong>11. </strong>Discard the column and store the purified plasmid DNA at -20°C.
 +
</p>
 +
</div>
 +
<!-- TO HERE - CUSTOM CONTENT HOLDER -->
 +
 +
<!-- FOR NEW ONE COPY FROM HERE - CUSTOM CONTENT HOLDER -->
 +
<div class="contentHolder" id="PROJECTPROTOCOLS">
 +
<h1>Miniprep Plasmid Purification</h1>
 +
<p>
 +
<strong>1. </strong>Resuspend the pelleted cells in 250 uL of the Resuspension Solution. Transfer the cell suspension to a microcentrifuge tube. The bacteria should be resuspended completely by vortexing or pipetting up and down until no cell clumps remain.<br>
 +
<strong>2. </strong>Add 250 uL of the Lysis Solution and mix thoroughly by inverting the tube 4-6 times until the solution becomes viscous and slightly clear. Do not vortex to avoid shearing of chromosomal DNA. Do not incubate for more than 5 min to avoid denaturation of supercoiled plasmid DNA.<br>
 +
<strong>3. </strong>Add 350 uL of the Neutralization Solution and mix immediately and thoroughly by inverting the tube 4-6 times. The neutralized bacterial lysate should become cloudy.<br>
 +
<strong>4. </strong>Centrifuge for 5 min to pellet cell debris and chromosomal DNA.<br>
 +
<strong>5. </strong>Transfer the supernatant to the supplied GeneJET spin column by decanting or pipetting. Avoid disturbing or transferring the white precipitate.<br>
 +
<strong>6. </strong>Centrifuge for 1 min. Discard the flow-through and place the column back into the same collection tube.<br>
 +
<strong>7. </strong>Add 500 uL of the Wash Solution to the GeneJET spin column. Centrifuge for 30-60 seconds and discard the flow-through. Place the column back into the same collection tube.<br>
 +
<strong>8. </strong>Repeat the wash procedure using 500 uL of the Wash Solution.<br>
 +
<strong>9. </strong>Discard the flow-through and centrifuge for an additional 1 min to remove residual Wash Solution. This step is essential to avoid residual ethanol in plasmid preps.<br>
 +
<strong>10. </strong>Transfer the GeneJET spin column into a fresh 1.5 mL microcentrifuge tube (not included). Add 50 uL of the Elution Buffer to the center of GeneJET spin column membrane to elute the plasmid DNA. Take care not to contact the membrane with the pipette tip. Incubate for 2 min at room temperature and centrifuge for 2 min.. For elution of plasmids or cosmids >20 kb, prewarm Elution Buffer to 70°C before applying to silica membrane.<br>
 +
<strong>11. </strong>Discard the column and store the purified plasmid DNA at -20°C.
 +
</p>
 +
</div>
 +
<!-- TO HERE - CUSTOM CONTENT HOLDER -->
 +
 +
<!-- FOR NEW ONE COPY FROM HERE - CUSTOM CONTENT HOLDER -->
 +
<div class="contentHolder" id="PROJECTPROTOCOLS">
 +
<h1>Miniprep Plasmid Purification</h1>
 +
<p>
 +
<strong>1. </strong>Resuspend the pelleted cells in 250 uL of the Resuspension Solution. Transfer the cell suspension to a microcentrifuge tube. The bacteria should be resuspended completely by vortexing or pipetting up and down until no cell clumps remain.<br>
 +
<strong>2. </strong>Add 250 uL of the Lysis Solution and mix thoroughly by inverting the tube 4-6 times until the solution becomes viscous and slightly clear. Do not vortex to avoid shearing of chromosomal DNA. Do not incubate for more than 5 min to avoid denaturation of supercoiled plasmid DNA.<br>
 +
<strong>3. </strong>Add 350 uL of the Neutralization Solution and mix immediately and thoroughly by inverting the tube 4-6 times. The neutralized bacterial lysate should become cloudy.<br>
 +
<strong>4. </strong>Centrifuge for 5 min to pellet cell debris and chromosomal DNA.<br>
 +
<strong>5. </strong>Transfer the supernatant to the supplied GeneJET spin column by decanting or pipetting. Avoid disturbing or transferring the white precipitate.<br>
 +
<strong>6. </strong>Centrifuge for 1 min. Discard the flow-through and place the column back into the same collection tube.<br>
 +
<strong>7. </strong>Add 500 uL of the Wash Solution to the GeneJET spin column. Centrifuge for 30-60 seconds and discard the flow-through. Place the column back into the same collection tube.<br>
 +
<strong>8. </strong>Repeat the wash procedure using 500 uL of the Wash Solution.<br>
 +
<strong>9. </strong>Discard the flow-through and centrifuge for an additional 1 min to remove residual Wash Solution. This step is essential to avoid residual ethanol in plasmid preps.<br>
 +
<strong>10. </strong>Transfer the GeneJET spin column into a fresh 1.5 mL microcentrifuge tube (not included). Add 50 uL of the Elution Buffer to the center of GeneJET spin column membrane to elute the plasmid DNA. Take care not to contact the membrane with the pipette tip. Incubate for 2 min at room temperature and centrifuge for 2 min.. For elution of plasmids or cosmids >20 kb, prewarm Elution Buffer to 70°C before applying to silica membrane.<br>
 +
<strong>11. </strong>Discard the column and store the purified plasmid DNA at -20°C.
 +
</p>
 +
</div>
 +
<!-- TO HERE - CUSTOM CONTENT HOLDER -->
 +
 +
<!-- FOR NEW ONE COPY FROM HERE - CUSTOM CONTENT HOLDER -->
 +
<div class="contentHolder" id="PROJECTPROTOCOLS">
 +
<h1>Miniprep Plasmid Purification</h1>
 +
<p>
 +
<strong>1. </strong>Resuspend the pelleted cells in 250 uL of the Resuspension Solution. Transfer the cell suspension to a microcentrifuge tube. The bacteria should be resuspended completely by vortexing or pipetting up and down until no cell clumps remain.<br>
 +
<strong>2. </strong>Add 250 uL of the Lysis Solution and mix thoroughly by inverting the tube 4-6 times until the solution becomes viscous and slightly clear. Do not vortex to avoid shearing of chromosomal DNA. Do not incubate for more than 5 min to avoid denaturation of supercoiled plasmid DNA.<br>
 +
<strong>3. </strong>Add 350 uL of the Neutralization Solution and mix immediately and thoroughly by inverting the tube 4-6 times. The neutralized bacterial lysate should become cloudy.<br>
 +
<strong>4. </strong>Centrifuge for 5 min to pellet cell debris and chromosomal DNA.<br>
 +
<strong>5. </strong>Transfer the supernatant to the supplied GeneJET spin column by decanting or pipetting. Avoid disturbing or transferring the white precipitate.<br>
 +
<strong>6. </strong>Centrifuge for 1 min. Discard the flow-through and place the column back into the same collection tube.<br>
 +
<strong>7. </strong>Add 500 uL of the Wash Solution to the GeneJET spin column. Centrifuge for 30-60 seconds and discard the flow-through. Place the column back into the same collection tube.<br>
 +
<strong>8. </strong>Repeat the wash procedure using 500 uL of the Wash Solution.<br>
 +
<strong>9. </strong>Discard the flow-through and centrifuge for an additional 1 min to remove residual Wash Solution. This step is essential to avoid residual ethanol in plasmid preps.<br>
 +
<strong>10. </strong>Transfer the GeneJET spin column into a fresh 1.5 mL microcentrifuge tube (not included). Add 50 uL of the Elution Buffer to the center of GeneJET spin column membrane to elute the plasmid DNA. Take care not to contact the membrane with the pipette tip. Incubate for 2 min at room temperature and centrifuge for 2 min.. For elution of plasmids or cosmids >20 kb, prewarm Elution Buffer to 70°C before applying to silica membrane.<br>
 +
<strong>11. </strong>Discard the column and store the purified plasmid DNA at -20°C.
 +
</p>
 +
</div>
 +
<!-- TO HERE - CUSTOM CONTENT HOLDER -->
 +
 +
<!-- FOR NEW ONE COPY FROM HERE - CUSTOM CONTENT HOLDER -->
 +
<div class="contentHolder" id="PROJECTPROTOCOLS">
 +
<h1>Miniprep Plasmid Purification</h1>
 +
<p>
 +
<strong>1. </strong>Resuspend the pelleted cells in 250 uL of the Resuspension Solution. Transfer the cell suspension to a microcentrifuge tube. The bacteria should be resuspended completely by vortexing or pipetting up and down until no cell clumps remain.<br>
 +
<strong>2. </strong>Add 250 uL of the Lysis Solution and mix thoroughly by inverting the tube 4-6 times until the solution becomes viscous and slightly clear. Do not vortex to avoid shearing of chromosomal DNA. Do not incubate for more than 5 min to avoid denaturation of supercoiled plasmid DNA.<br>
 +
<strong>3. </strong>Add 350 uL of the Neutralization Solution and mix immediately and thoroughly by inverting the tube 4-6 times. The neutralized bacterial lysate should become cloudy.<br>
 +
<strong>4. </strong>Centrifuge for 5 min to pellet cell debris and chromosomal DNA.<br>
 +
<strong>5. </strong>Transfer the supernatant to the supplied GeneJET spin column by decanting or pipetting. Avoid disturbing or transferring the white precipitate.<br>
 +
<strong>6. </strong>Centrifuge for 1 min. Discard the flow-through and place the column back into the same collection tube.<br>
 +
<strong>7. </strong>Add 500 uL of the Wash Solution to the GeneJET spin column. Centrifuge for 30-60 seconds and discard the flow-through. Place the column back into the same collection tube.<br>
 +
<strong>8. </strong>Repeat the wash procedure using 500 uL of the Wash Solution.<br>
 +
<strong>9. </strong>Discard the flow-through and centrifuge for an additional 1 min to remove residual Wash Solution. This step is essential to avoid residual ethanol in plasmid preps.<br>
 +
<strong>10. </strong>Transfer the GeneJET spin column into a fresh 1.5 mL microcentrifuge tube (not included). Add 50 uL of the Elution Buffer to the center of GeneJET spin column membrane to elute the plasmid DNA. Take care not to contact the membrane with the pipette tip. Incubate for 2 min at room temperature and centrifuge for 2 min.. For elution of plasmids or cosmids >20 kb, prewarm Elution Buffer to 70°C before applying to silica membrane.<br>
 +
<strong>11. </strong>Discard the column and store the purified plasmid DNA at -20°C.
 +
</p>
 +
</div>
 +
<!-- TO HERE - CUSTOM CONTENT HOLDER -->
 +
  
  

Revision as of 20:46, 17 October 2018

PROJECT USE IS IT SAFE USED ORGANISM STRAINS POTENTIAL RISKS WASTE TREATMENT EMERGENCY REACTIONS PROTECTIVE EQUIPMENT OTHER RULES

Miniprep Plasmid Purification

1. Resuspend the pelleted cells in 250 uL of the Resuspension Solution. Transfer the cell suspension to a microcentrifuge tube. The bacteria should be resuspended completely by vortexing or pipetting up and down until no cell clumps remain.
2. Add 250 uL of the Lysis Solution and mix thoroughly by inverting the tube 4-6 times until the solution becomes viscous and slightly clear. Do not vortex to avoid shearing of chromosomal DNA. Do not incubate for more than 5 min to avoid denaturation of supercoiled plasmid DNA.
3. Add 350 uL of the Neutralization Solution and mix immediately and thoroughly by inverting the tube 4-6 times. The neutralized bacterial lysate should become cloudy.
4. Centrifuge for 5 min to pellet cell debris and chromosomal DNA.
5. Transfer the supernatant to the supplied GeneJET spin column by decanting or pipetting. Avoid disturbing or transferring the white precipitate.
6. Centrifuge for 1 min. Discard the flow-through and place the column back into the same collection tube.
7. Add 500 uL of the Wash Solution to the GeneJET spin column. Centrifuge for 30-60 seconds and discard the flow-through. Place the column back into the same collection tube.
8. Repeat the wash procedure using 500 uL of the Wash Solution.
9. Discard the flow-through and centrifuge for an additional 1 min to remove residual Wash Solution. This step is essential to avoid residual ethanol in plasmid preps.
10. Transfer the GeneJET spin column into a fresh 1.5 mL microcentrifuge tube (not included). Add 50 uL of the Elution Buffer to the center of GeneJET spin column membrane to elute the plasmid DNA. Take care not to contact the membrane with the pipette tip. Incubate for 2 min at room temperature and centrifuge for 2 min.. For elution of plasmids or cosmids >20 kb, prewarm Elution Buffer to 70°C before applying to silica membrane.
11. Discard the column and store the purified plasmid DNA at -20°C.

PureLink® Quick Gel Extraction and PCR Purification Combo Kit

Purifying PCR fragments or reaction clean-up

1. Combine. Add 4 volumes of Binding Buffer (B2) with isopropanol to 1 volume of a PCR sample (50–100 uL). Mix well.
2. Load. Pipet the sample into a PureLink® Clean-up Spin Column in a Wash Tube. Centrifuge the column at >10,000 × g for 1 minute. Discard the flow-through.
3. Wash. Re-insert the column into the Wash Tube and add 650 uL Wash Buffer (W1) with ethanol. Centrifuge the column at >10,000 × g for 1 minute.
4. Remove ethanol. Discard the flow-through and place the column in the same Wash Tube. Centrifuge the column at maximum speed for 2–3 minutes.
5. Elute. Place the column into a clean 1.5-mL Elution Tube. Add 50 uL Elution Buffer (E1) to the column. Incubate the column at room temperature for 1 minute. Centrifuge the column at maximum speed for 1 minute.
6. Store. The elution tube contains the purified PCR product. Store the purified DNA at 4°C for immediate use or at -20°C for long-term storage.

Miniprep Plasmid Purification

1. Resuspend the pelleted cells in 250 uL of the Resuspension Solution. Transfer the cell suspension to a microcentrifuge tube. The bacteria should be resuspended completely by vortexing or pipetting up and down until no cell clumps remain.
2. Add 250 uL of the Lysis Solution and mix thoroughly by inverting the tube 4-6 times until the solution becomes viscous and slightly clear. Do not vortex to avoid shearing of chromosomal DNA. Do not incubate for more than 5 min to avoid denaturation of supercoiled plasmid DNA.
3. Add 350 uL of the Neutralization Solution and mix immediately and thoroughly by inverting the tube 4-6 times. The neutralized bacterial lysate should become cloudy.
4. Centrifuge for 5 min to pellet cell debris and chromosomal DNA.
5. Transfer the supernatant to the supplied GeneJET spin column by decanting or pipetting. Avoid disturbing or transferring the white precipitate.
6. Centrifuge for 1 min. Discard the flow-through and place the column back into the same collection tube.
7. Add 500 uL of the Wash Solution to the GeneJET spin column. Centrifuge for 30-60 seconds and discard the flow-through. Place the column back into the same collection tube.
8. Repeat the wash procedure using 500 uL of the Wash Solution.
9. Discard the flow-through and centrifuge for an additional 1 min to remove residual Wash Solution. This step is essential to avoid residual ethanol in plasmid preps.
10. Transfer the GeneJET spin column into a fresh 1.5 mL microcentrifuge tube (not included). Add 50 uL of the Elution Buffer to the center of GeneJET spin column membrane to elute the plasmid DNA. Take care not to contact the membrane with the pipette tip. Incubate for 2 min at room temperature and centrifuge for 2 min.. For elution of plasmids or cosmids >20 kb, prewarm Elution Buffer to 70°C before applying to silica membrane.
11. Discard the column and store the purified plasmid DNA at -20°C.

Miniprep Plasmid Purification

1. Resuspend the pelleted cells in 250 uL of the Resuspension Solution. Transfer the cell suspension to a microcentrifuge tube. The bacteria should be resuspended completely by vortexing or pipetting up and down until no cell clumps remain.
2. Add 250 uL of the Lysis Solution and mix thoroughly by inverting the tube 4-6 times until the solution becomes viscous and slightly clear. Do not vortex to avoid shearing of chromosomal DNA. Do not incubate for more than 5 min to avoid denaturation of supercoiled plasmid DNA.
3. Add 350 uL of the Neutralization Solution and mix immediately and thoroughly by inverting the tube 4-6 times. The neutralized bacterial lysate should become cloudy.
4. Centrifuge for 5 min to pellet cell debris and chromosomal DNA.
5. Transfer the supernatant to the supplied GeneJET spin column by decanting or pipetting. Avoid disturbing or transferring the white precipitate.
6. Centrifuge for 1 min. Discard the flow-through and place the column back into the same collection tube.
7. Add 500 uL of the Wash Solution to the GeneJET spin column. Centrifuge for 30-60 seconds and discard the flow-through. Place the column back into the same collection tube.
8. Repeat the wash procedure using 500 uL of the Wash Solution.
9. Discard the flow-through and centrifuge for an additional 1 min to remove residual Wash Solution. This step is essential to avoid residual ethanol in plasmid preps.
10. Transfer the GeneJET spin column into a fresh 1.5 mL microcentrifuge tube (not included). Add 50 uL of the Elution Buffer to the center of GeneJET spin column membrane to elute the plasmid DNA. Take care not to contact the membrane with the pipette tip. Incubate for 2 min at room temperature and centrifuge for 2 min.. For elution of plasmids or cosmids >20 kb, prewarm Elution Buffer to 70°C before applying to silica membrane.
11. Discard the column and store the purified plasmid DNA at -20°C.

Miniprep Plasmid Purification

1. Resuspend the pelleted cells in 250 uL of the Resuspension Solution. Transfer the cell suspension to a microcentrifuge tube. The bacteria should be resuspended completely by vortexing or pipetting up and down until no cell clumps remain.
2. Add 250 uL of the Lysis Solution and mix thoroughly by inverting the tube 4-6 times until the solution becomes viscous and slightly clear. Do not vortex to avoid shearing of chromosomal DNA. Do not incubate for more than 5 min to avoid denaturation of supercoiled plasmid DNA.
3. Add 350 uL of the Neutralization Solution and mix immediately and thoroughly by inverting the tube 4-6 times. The neutralized bacterial lysate should become cloudy.
4. Centrifuge for 5 min to pellet cell debris and chromosomal DNA.
5. Transfer the supernatant to the supplied GeneJET spin column by decanting or pipetting. Avoid disturbing or transferring the white precipitate.
6. Centrifuge for 1 min. Discard the flow-through and place the column back into the same collection tube.
7. Add 500 uL of the Wash Solution to the GeneJET spin column. Centrifuge for 30-60 seconds and discard the flow-through. Place the column back into the same collection tube.
8. Repeat the wash procedure using 500 uL of the Wash Solution.
9. Discard the flow-through and centrifuge for an additional 1 min to remove residual Wash Solution. This step is essential to avoid residual ethanol in plasmid preps.
10. Transfer the GeneJET spin column into a fresh 1.5 mL microcentrifuge tube (not included). Add 50 uL of the Elution Buffer to the center of GeneJET spin column membrane to elute the plasmid DNA. Take care not to contact the membrane with the pipette tip. Incubate for 2 min at room temperature and centrifuge for 2 min.. For elution of plasmids or cosmids >20 kb, prewarm Elution Buffer to 70°C before applying to silica membrane.
11. Discard the column and store the purified plasmid DNA at -20°C.

Miniprep Plasmid Purification

1. Resuspend the pelleted cells in 250 uL of the Resuspension Solution. Transfer the cell suspension to a microcentrifuge tube. The bacteria should be resuspended completely by vortexing or pipetting up and down until no cell clumps remain.
2. Add 250 uL of the Lysis Solution and mix thoroughly by inverting the tube 4-6 times until the solution becomes viscous and slightly clear. Do not vortex to avoid shearing of chromosomal DNA. Do not incubate for more than 5 min to avoid denaturation of supercoiled plasmid DNA.
3. Add 350 uL of the Neutralization Solution and mix immediately and thoroughly by inverting the tube 4-6 times. The neutralized bacterial lysate should become cloudy.
4. Centrifuge for 5 min to pellet cell debris and chromosomal DNA.
5. Transfer the supernatant to the supplied GeneJET spin column by decanting or pipetting. Avoid disturbing or transferring the white precipitate.
6. Centrifuge for 1 min. Discard the flow-through and place the column back into the same collection tube.
7. Add 500 uL of the Wash Solution to the GeneJET spin column. Centrifuge for 30-60 seconds and discard the flow-through. Place the column back into the same collection tube.
8. Repeat the wash procedure using 500 uL of the Wash Solution.
9. Discard the flow-through and centrifuge for an additional 1 min to remove residual Wash Solution. This step is essential to avoid residual ethanol in plasmid preps.
10. Transfer the GeneJET spin column into a fresh 1.5 mL microcentrifuge tube (not included). Add 50 uL of the Elution Buffer to the center of GeneJET spin column membrane to elute the plasmid DNA. Take care not to contact the membrane with the pipette tip. Incubate for 2 min at room temperature and centrifuge for 2 min.. For elution of plasmids or cosmids >20 kb, prewarm Elution Buffer to 70°C before applying to silica membrane.
11. Discard the column and store the purified plasmid DNA at -20°C.

Miniprep Plasmid Purification

1. Resuspend the pelleted cells in 250 uL of the Resuspension Solution. Transfer the cell suspension to a microcentrifuge tube. The bacteria should be resuspended completely by vortexing or pipetting up and down until no cell clumps remain.
2. Add 250 uL of the Lysis Solution and mix thoroughly by inverting the tube 4-6 times until the solution becomes viscous and slightly clear. Do not vortex to avoid shearing of chromosomal DNA. Do not incubate for more than 5 min to avoid denaturation of supercoiled plasmid DNA.
3. Add 350 uL of the Neutralization Solution and mix immediately and thoroughly by inverting the tube 4-6 times. The neutralized bacterial lysate should become cloudy.
4. Centrifuge for 5 min to pellet cell debris and chromosomal DNA.
5. Transfer the supernatant to the supplied GeneJET spin column by decanting or pipetting. Avoid disturbing or transferring the white precipitate.
6. Centrifuge for 1 min. Discard the flow-through and place the column back into the same collection tube.
7. Add 500 uL of the Wash Solution to the GeneJET spin column. Centrifuge for 30-60 seconds and discard the flow-through. Place the column back into the same collection tube.
8. Repeat the wash procedure using 500 uL of the Wash Solution.
9. Discard the flow-through and centrifuge for an additional 1 min to remove residual Wash Solution. This step is essential to avoid residual ethanol in plasmid preps.
10. Transfer the GeneJET spin column into a fresh 1.5 mL microcentrifuge tube (not included). Add 50 uL of the Elution Buffer to the center of GeneJET spin column membrane to elute the plasmid DNA. Take care not to contact the membrane with the pipette tip. Incubate for 2 min at room temperature and centrifuge for 2 min.. For elution of plasmids or cosmids >20 kb, prewarm Elution Buffer to 70°C before applying to silica membrane.
11. Discard the column and store the purified plasmid DNA at -20°C.