Isolation of DNA from agarose gel: Difference between revisions
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{{Protokoll-Mix|Inhalt= | {{Protokoll-Mix|Inhalt= | ||
„QIAquick gel extraction“-Kit <br>Balance<br>2 ml Eppendorf tubes <br> | |||
Heating block at 50° C <br>Vortex<br>3M NaCOOH pH 5.0 (optional) <br>Isopropanol (optional) <br>Fragment, isolated from the gel<br>}} | |||
== Procedure == | |||
* For 100 mg gel add 100 µl buffer QG | |||
* Incubate at 50° C until the gel is completely solved (vortex every 2-3 minutes) | |||
* The colour of the solution after solving should be yellow (like the colour of QG-buffer) | |||
* In case of orange or violet colour titrate back to yellow colour with NaCOOH. | |||
* For fragments smaller than 500bp or bigger than 4kbp add 100 µl isopropanol for each 100 mg gel | |||
* Apply 750 µl solution to the QIAprep spin column | |||
* Centrifuge in a table-top microcentrifuge (1 min, 10,000 rpm) and discard the flow through ('''keep the spin column''') | |||
* Repeat applying of the sample on the same column in case of bigger volumes | |||
* Wash QIAprep spin column with 750 µl PE buffer, centrifuge and discard the flow-through | |||
* Centrifuge for an additional 1 min to remove residual wash buffer (important) | |||
* Place the spin column into a clean 1,5 ml Eppendorf tube | |||
* Add to the center of each spin column 40 µl ddH<sub>2</sub>0 | |||
* Let stand for 2 minutes (RT) | |||
* Centrifuge for 1 min and collect the isolated DNA into the Eppendorf tube | |||
Latest revision as of 11:25, 30 May 2025
Materials
„QIAquick gel extraction“-Kit
Balance
2 ml Eppendorf tubes
Heating block at 50° C
Vortex
3M NaCOOH pH 5.0 (optional)
Isopropanol (optional)
Fragment, isolated from the gel
Balance
2 ml Eppendorf tubes
Heating block at 50° C
Vortex
3M NaCOOH pH 5.0 (optional)
Isopropanol (optional)
Fragment, isolated from the gel
Procedure
- For 100 mg gel add 100 µl buffer QG
- Incubate at 50° C until the gel is completely solved (vortex every 2-3 minutes)
- The colour of the solution after solving should be yellow (like the colour of QG-buffer)
- In case of orange or violet colour titrate back to yellow colour with NaCOOH.
- For fragments smaller than 500bp or bigger than 4kbp add 100 µl isopropanol for each 100 mg gel
- Apply 750 µl solution to the QIAprep spin column
- Centrifuge in a table-top microcentrifuge (1 min, 10,000 rpm) and discard the flow through (keep the spin column)
- Repeat applying of the sample on the same column in case of bigger volumes
- Wash QIAprep spin column with 750 µl PE buffer, centrifuge and discard the flow-through
- Centrifuge for an additional 1 min to remove residual wash buffer (important)
- Place the spin column into a clean 1,5 ml Eppendorf tube
- Add to the center of each spin column 40 µl ddH20
- Let stand for 2 minutes (RT)
- Centrifuge for 1 min and collect the isolated DNA into the Eppendorf tube