Isolation of DNA from agarose gel

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

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