Difference between revisions of "Team:KU Leuven/Research/Methods"
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<dd>11. Transfer the GeneJET spin column to a fresh 1.5 mL microcentrifuge tube. Add 50 µL of the Elution Buffer to the center of the GeneJET spin column membrane to elute the plasmid DNA. Take care not to touch the membrane with the pipette tip. Incubate for 2 min at room temperature and centrifuge for 2 min. <br/> | <dd>11. Transfer the GeneJET spin column to a fresh 1.5 mL microcentrifuge tube. Add 50 µL of the Elution Buffer to the center of the GeneJET spin column membrane to elute the plasmid DNA. Take care not to touch the membrane with the pipette tip. Incubate for 2 min at room temperature and centrifuge for 2 min. <br/> | ||
<br/> | <br/> | ||
− | <b>Note. </b> An additional elution step (optional) with Elution Buffer or water will recover residual DNA from the membrane and increase the overall yield by 10-20%. For elution of plasmids or cosmids >20 kb, prewarm Elution Buffer to 70°C before applying to silica membrane. </dd> | + | <b>Note.</b> An additional elution step (optional) with Elution Buffer or water will recover residual DNA from the membrane and increase the overall yield by 10-20%. For elution of plasmids or cosmids >20 kb, prewarm Elution Buffer to 70°C before applying to silica membrane. </dd> |
<dd>12. Discard the column and store the purified plasmid DNA at -20°C. </dd> | <dd>12. Discard the column and store the purified plasmid DNA at -20°C. </dd> | ||
</dl> | </dl> | ||
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<p><b>Protocol:</b></p> | <p><b>Protocol:</b></p> | ||
<dl> | <dl> | ||
− | <dd>1. Add a 1:1 volume of Binding Buffer to completed PCR mixture | + | <dd>1. Add a 1:1 volume of Binding Buffer to completed PCR mixture and mix thoroughly. Check the color of the solution. A yellow color indicates an optimal pH for DNA binding. If the color of the solution is orange or violet, add 10 µL of 3 M sodium acetate, pH 5.2 solution and mix. The color of the mix will become yellow.</dd> |
− | <dd> 2. Transfer up to 800 µL of the solution from step 1 | + | <dd>2. Transfer up to 800 µL of the solution from step 1 to the GeneJET purification column. Centrifuge for 30-60 s. Discard the flow-through. <br/> |
− | <dd>3. Add 700 µL of Wash Buffer | + | <br/> |
− | <dd>4. Transfer the GeneJET purification column to a clean 1.5 mL microcentrifuge tube | + | <b>Note</b>. If the total volume exceeds 800 µL, the solution can be added to the column in stages. After the addition of 800 µL of solution, centrifuge the column for 30-60 s and discard flow-through. Repeat until the entire solution has been added to the column membrane. Close the bag with GeneJET Purification Columns tightly after each use! </dd> |
+ | <dd>3. Add 700 µL of Wash Buffer to the GeneJET purification column. Centrifuge for 30-60 s. Discard the flow-through and place the purification column back into the collection tube.</dd> | ||
+ | <dd>4. Transfer the GeneJET purification column to a clean 1.5 mL microcentrifuge tube, add 50 µL of Elution Buffer to the center of the GeneJET purification column membrane and centrifuge for 1 min.</dd> | ||
<p><b>Note.</b> | <p><b>Note.</b> | ||
For low DNA amounts the elution volumes can be reduced to increase DNA concentration. An elution volume between 20-50 µL does not significantly reduce the DNA yield. However, elution volumes less than 10 µL 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 µL and DNA amount is ≥5 µg, incubate column for 1 min at room temperature before centrifugation.<p></dd> | For low DNA amounts the elution volumes can be reduced to increase DNA concentration. An elution volume between 20-50 µL does not significantly reduce the DNA yield. However, elution volumes less than 10 µL 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 µL and DNA amount is ≥5 µg, incubate column for 1 min at room temperature before centrifugation.<p></dd> |
Revision as of 13:45, 16 September 2015
Methods
On this page you can find all of the methods and protocols used in the lab to obtain our results. For some techniques, we included some basic theory, since it is a prerequisite to get acquainted with the theory behind these techniques before using them. To learn more about them, click the titles below!
Contact
Address: Celestijnenlaan 200G room 00.08 - 3001 Heverlee
Telephone: +32(0)16 32 73 19
Email: igem@chem.kuleuven.be