51 PCR Clean-Up
To remove the reagents from the PCR and isolate pure DNA for Sanger sequencing, we need to “clean up” our amplified DNA samples. In BIT 295, we use reagents from the QIAGEN DNA Clean-Up Kit.
- Add all 50 µL of the PCR reaction to five volumes (250 µL) PB buffer
- Mix and transfer to pink spin column
- Centrifuge at 10,000 rpm for 30 sec
- Remove flow through
- Add 750 µL PE to wash the column. Incubate at RT for 1 minute.
- Centrifuge at 10,000 rpm for 30 sec
- Remove the spin column from the collection tube and add to a new wash tube
- Centrifuge at 10,000 rpm for 30 sec to remove all ethanol wash
- Remove the spin column from the collection tube and add to a new collection tube
- Add 50 µL EB to elute. Incubate at RT for 1 minute.
- Centrifuge at 10,000 rpm for 30 sec
- Remove the spin column and throw away
- Quantify 2 µl using the Implen that has been blanked with the elution buffer (EB).
- DNA can be stored at 4°C or on ice while being used and should be stored long-term at -20°C.
