PureLink™ Genomisches DNA-Minikit

We would recommend using our PureLink gDNA Mini Kit or our ChargeSwitch gDNA Mini Bacteria Kit (Cat. No. CS11301), which can isolate both gram-positive and gram-negative bacteria. No centrifugation or filtration steps are necessary using this simple one-tube protocol. Read more about bacterial DNA extraction here (http://www.thermofisher.com/us/en/home/life-science/dna-rna-purification-analysis/dna-extraction/genomic-dna-extraction/bacteria-dna-extraction.html).

We offer TRIzol reagent that will allow isolation of DNA and RNA from the same sample. Alternatively, we have the following method that has been validated by our R&D team; for sequential isolation of gDNA and total RNA from the same sample. This method involves using 2 of our kits: 1) PureLink RNA Mini Kit (Cat. No. 12183018A, 12183020, 12183025) and 2) PureLink Genomic DNA Mini Kit (Cat. No. K182002, K182000, K182001).

The protocol is detailed below:

Before starting:
- Label all spin columns and buffers from each kit with kit names to prevent confusion.
- Prepare lysis buffer and wash buffers according to the protocol from each kit.
1. Preparing lysates:
- Add 300 µL of lysis buffer (from Purelink RNA Mini Kit, beta-mercaptoethanol added) to cell or tissue sample, lyse the cells as recommended.

2. DNA isolation:
- Load all of the lysate directly onto a Purelink gDNA column (from PureLink Genomic DNA Mini Kit), save flow-through for RNA isolation.
- Centrifuge the Purelink gDNA column at 10,000 x g for 1 min.
- Wash the Purelink gDNA column with 500 µL of Wash Buffer 1 (from PureLink Genomic DNA Mini Kit, ethanol added), centrifuge at 10,000 x g for 1 min.
- Add 500 µL of Wash Buffer 2 (from PureLink Genomic DNA Mini Kit, ethanol added), centrifuge at maximum speed for 3 min to dry the membrane.
- Add 100 µL of Elution Buffer (from PureLink Genomic DNA Mini Kit), incubate at room temperature for 1 min and centrifuge at 10,000 x g for 1 min (yield can be increased if an optional second elution step is added).
- This is purified gDNA.

3. RNA isolation:
- To the above saved flow-through, add same volume of 70% ethanol, mix well and load the lysate/ethanol mix (including all precipitates) onto an RNA spin cartridge (from Purelink RNA Mini Kit).
- Centrifuge at 12,000 x g for 15 sec. Discard flow-through.
- Wash the RNA spin cartridge with 700 µL of Wash Buffer 1 (from Purelink RNA Mini Kit, ethanol added), centrifuge at 12,000 x g for 15 sec.
- Wash twice with 500 µL of Wash Buffer 2 (from Purelink RNA Mini Kit, ethanol added). After the second wash, centrifuge at 12,000 x g for 1 min to dry the membrane.
- Add 50 µL of RNase-free water onto the RNA spin cartridge, incubate at room temperature for 1 min and centrifuge at 12,000 x g for 2 min (yield can be increased if an optional second elution step is added).
- This is purified RNA.

We recommend the following DNA isolation kits:

- RecoverAll Total Nucleic Acid Isolation Kit for FFPE (Cat. No. AM1975)
- Ion Ampliseq Direct FFPE DNA Kit (Cat. Nos. A31133, A31136)
- MagMAX FFPE DNA/RNA Ultra Kit (Cat. No. A31881)
- PureLink Genomic DNA Mini Kit (Cat. Nos, K182000, K182001, K182002)