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Team:METU Turkey/Results Discussion/PCR Purification
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Cerenseref (Talk | contribs) (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>MATERIALS | <br>MATERIALS | ||
<br>- Heating block | <br>- Heating block | ||
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<br>SOLUTIONS | <br>SOLUTIONS | ||
| - | <br>- Prior to the initial use of the PCR Purification Kit, dilute the Wash Buffer (concentrated) with ethanol (96- | + | <br>- Prior to the initial use of the PCR Purification Kit, dilute the Wash Buffer (concentrated) with ethanol (96-100%) |
| - | <br>- Examine the Binding Buffer for precipitates before each use. Dissolve the precipitate by warming the solution | + | <br>- Examine the Binding Buffer for precipitates before each use. Dissolve the precipitate by warming the solution to |
| - | + | 37 C and cooling to 25 C. | |
<br>- Wear gloves when handling the Binding Buffer | <br>- Wear gloves when handling the Binding Buffer | ||
| - | <br>- Check the color of the Binding Buffer solution. A yellow color indicates an optimal pH for DNA binding. If the | + | <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 |
| - | + | acetate, pH 5.2 solution and mix. The color of the mix will become yellow. | |
<br>CHECK-LIST PROCEDURE | <br>CHECK-LIST PROCEDURE | ||
<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. | ||
| - | <br>- If the DNA fragment is <500 bp, add a 1:2 volume of 100 % isopropanol [optional]. Mix by pipetting gently up | + | <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. | ||
| - | + | [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. | ||
| - | <br>- Centrifuge the empty purification column for an additional 1 min at >12000 g to completely remove any residual | + | <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. | ||
| - | <br>- Add 50 uL of Elution Buffer to the center of the purification column membrane and centrifuge for 1 min at | + | <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. | ||
<br>TROUBLESHOOTING | <br>TROUBLESHOOTING | ||
| - | <br>- For low DNA amounts the elution volumes can be reduced to increase DNA concentration. An elution volume between | + | <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 |
| - | + | 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. | ||
| - | <br>- If the elution volume is 10 uL and DNA amount is >5 ug, incubate column for 1 min at room temperature before | + | <br>- If the elution volume is 10 uL and DNA amount is >5 ug, incubate column for 1 min at room temperature before centrifugation. |
Revision as of 20:38, 27 October 2010
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.
