Team:METU Turkey/Results Discussion/PCR Purification

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(New page: <br>MATERIALS <br>- Heating block <br>- Microcentrifuge <br>- PCR Purification Kit <br>- Binding Buffer <br>- Wash Buffer <br>- Elution Buffer <br>- Water nuclease free ...)
 
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<br><h3>MATERIALS</h3>        
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<br>MATERIALS       
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<br>- Heating block
<br>- Heating block
<br>- Microcentrifuge
<br>- Microcentrifuge
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<br>- 3 M sodium acetate, pH 5.2
<br>- 3 M sodium acetate, pH 5.2
   
   
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<br>SOLUTIONS     
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<br><h3>SOLUTIONS</h3>      
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<br>- Prior to the initial use of the PCR Purification Kit, dilute the Wash Buffer (concentrated) with ethanol (96-<br>100%)
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<br>- Prior to the initial use of the PCR Purification Kit, dilute the Wash Buffer (concentrated) with ethanol (96-100%)
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<br>- Examine the Binding Buffer for precipitates before each use. Dissolve the precipitate by warming the solution <br>to  
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<br>- Examine the Binding Buffer for precipitates before each use. Dissolve the precipitate by warming the solution to  
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<br>37 C and cooling to 25 C.
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37 C and cooling to 25 C.
<br>- Wear gloves when handling the Binding Buffer
<br>- Wear gloves when handling the Binding Buffer
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<br>- Check the color of the Binding Buffer solution. A yellow color indicates an optimal pH for DNA binding. If the <br>color of the solution is orange or violet, add 10 uL of 3 M sodium
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<br>- Check the color of the Binding Buffer solution. A yellow color indicates an optimal pH for DNA binding. If the color of the solution is orange or violet, add 10 uL of 3 M sodium
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<br>acetate, pH 5.2 solution and mix. The color of the mix will become yellow.
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acetate, pH 5.2 solution and mix. The color of the mix will become yellow.
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<br>CHECK-LIST PROCEDURE    
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<br><h3>CHECK-LIST PROCEDURE</h3>   
<br>- Add a 1:1 volume of Binding Buffer to completed PCR mixture. Mix by pipetting gently up and down.
<br>- Add a 1:1 volume of Binding Buffer to completed PCR mixture. Mix by pipetting gently up and down.
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<br>- If the DNA fragment is <500 bp, add a 1:2 volume of 100 % isopropanol [optional]. Mix by pipetting gently up <br>and down.
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<br>- If the DNA fragment is <500 bp, add a 1:2 volume of 100 % isopropanol [optional]. Mix by pipetting gently up and down.
<br>- Transfer up to 800 uL of the solution from epp to the purification column.  
<br>- Transfer up to 800 uL of the solution from epp to the purification column.  
<br>- Centrifuge for 30-60 sec at >12000 g.  
<br>- Centrifuge for 30-60 sec at >12000 g.  
<br>- Discard the supernatant from collection tube.
<br>- Discard the supernatant from collection tube.
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<br>[If the total volume exceeds 800 uL, the solution can be added to the column in stages. After the addition of 800 <br>uL of solution, centrifuge the column for 30-60 sec and discard flow through. Repeat until the entire solution <br>has been added to the column membrane.]
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[If the total volume exceeds 800 uL, the solution can be added to the column in stages. After the addition of 800 uL of solution, centrifuge the column for 30-60 sec and discard flow through. Repeat until the entire solution <br>has been added to the column membrane.]
<br>- Add 700 uL of Wash Buffer (diluted) to the purification column.  
<br>- Add 700 uL of Wash Buffer (diluted) to the purification column.  
<br>- Centrifuge for 30-60 sec at >12000 g.
<br>- Centrifuge for 30-60 sec at >12000 g.
<br>- Discard the flow through and place the purification column back into the collection tube.
<br>- Discard the flow through and place the purification column back into the collection tube.
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<br>- Centrifuge the empty purification column for an additional 1 min at >12000 g to completely remove any residual <br>wash buffer.
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<br>- Centrifuge the empty purification column for an additional 1 min at >12000 g to completely remove any residual wash buffer.
<br>- Transfer the purification column to a clean 1.5 mL microcentrifuge tube.
<br>- Transfer the purification column to a clean 1.5 mL microcentrifuge tube.
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<br>- Add 50 uL of Elution Buffer to the center of the purification column membrane and centrifuge for 1 min at <br>>12000 g.
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<br>- Add 50 uL of Elution Buffer to the center of the purification column membrane and centrifuge for 1 min at 12000 g.
<br>- Discard the purification column and store the purifi ed DNA at -20 C.
<br>- Discard the purification column and store the purifi ed DNA at -20 C.
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<br>TROUBLESHOOTING  
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<br><h3>TROUBLESHOOTING</h3>
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<br>- For low DNA amounts the elution volumes can be reduced to increase DNA concentration. An elution volume between <br>20-50 uL does not significantly reduce the
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<br>- For low DNA amounts the elution volumes can be reduced to increase DNA concentration. An elution volume between 20-50 uL does not significantly reduce the
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<br>DNA yield. However, elution volumes less than 10 uL are not recommended.
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DNA yield. However, elution volumes less than 10 uL are not recommended.
<br>- If DNA fragment is >10 kb, prewarm Elution Buffer to 65 C before applying to column.
<br>- If DNA fragment is >10 kb, prewarm Elution Buffer to 65 C before applying to column.
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<br>- If the elution volume is 10 uL and DNA amount is >5 ug, incubate column for 1 min at room temperature before <br>centrifugation.
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<br>- If the elution volume is 10 uL and DNA amount is >5 ug, incubate column for 1 min at room temperature before centrifugation.

Latest revision as of 03:49, 28 October 2010


Contents

MATERIALS


- Heating block
- Microcentrifuge


- PCR Purification Kit

  
- Binding Buffer
- Wash Buffer
- Elution Buffer


- Water nuclease free
- Purification columns
- 1.5 or 2 mL microcentrifuge tubes
- Ethanol 96-100 %.
- 3 M sodium acetate, pH 5.2


SOLUTIONS


- Prior to the initial use of the PCR Purification Kit, dilute the Wash Buffer (concentrated) with ethanol (96-100%)
- Examine the Binding Buffer for precipitates before each use. Dissolve the precipitate by warming the solution to 37 C and cooling to 25 C.
- Wear gloves when handling the Binding Buffer
- Check the color of the Binding Buffer solution. A yellow color indicates an optimal pH for DNA binding. If the color of the solution is orange or violet, add 10 uL of 3 M sodium acetate, pH 5.2 solution and mix. The color of the mix will become yellow.



CHECK-LIST PROCEDURE


- Add a 1:1 volume of Binding Buffer to completed PCR mixture. Mix by pipetting gently up and down.
- If the DNA fragment is <500 bp, add a 1:2 volume of 100 % isopropanol [optional]. Mix by pipetting gently up and down.
- Transfer up to 800 uL of the solution from epp to the purification column.
- Centrifuge for 30-60 sec at >12000 g.
- Discard the supernatant from collection tube. [If the total volume exceeds 800 uL, the solution can be added to the column in stages. After the addition of 800 uL of solution, centrifuge the column for 30-60 sec and discard flow through. Repeat until the entire solution
has been added to the column membrane.]
- Add 700 uL of Wash Buffer (diluted) to the purification column.
- Centrifuge for 30-60 sec at >12000 g.
- Discard the flow through and place the purification column back into the collection tube.
- Centrifuge the empty purification column for an additional 1 min at >12000 g to completely remove any residual wash buffer.
- Transfer the purification column to a clean 1.5 mL microcentrifuge tube.
- Add 50 uL of Elution Buffer to the center of the purification column membrane and centrifuge for 1 min at 12000 g.
- Discard the purification column and store the purifi ed DNA at -20 C.



TROUBLESHOOTING


- For low DNA amounts the elution volumes can be reduced to increase DNA concentration. An elution volume between 20-50 uL does not significantly reduce the DNA yield. However, elution volumes less than 10 uL are not recommended.
- If DNA fragment is >10 kb, prewarm Elution Buffer to 65 C before applying to column.
- If the elution volume is 10 uL and DNA amount is >5 ug, incubate column for 1 min at room temperature before centrifugation.