Team:METU Turkey/Results Discussion/Genomic DNA purification

From 2010.igem.org

CHECK-LIST PROCEDURE
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- Harvest up to 2x10^9 bacterial cells in a 1.5 or 2 mL microcentrifuge tube by centrifugation for 10 min at 5000 x g. Discard the supernatant.
- Resuspend the pellet in 180 uL of Digestion Solution. Add 20 uL of Proteinase K Solution and mix thoroughly by vortexing or pipetting to obtain a uniform suspension.
- Incubate the sample at 56C while vortexing occasionally or use a shaking water bath, rocking platform or thermomixer until the cells are completely lysed (∼30 min).
- Add 20 uL of RNase A Solution, mix by vortexing and incubate the mixture for 10 min at room temperature.
- Add 200 uL of Lysis Solution to the sample. Mix thoroughly by vortexing for about 15 sec until a homogeneous mixture is obtained.
- Add 400 uL of 50% ethanol and mix by pipetting or vortexing.
- Transfer the prepared lysate to a GeneJET™ Genomic DNA Purification Column inserted in a collection tube. Centrifuge the column for 1 min at 6000 x g. Discard the collection tube containing the flow-through solution. Place the GeneJET™ Genomic DNA Purification Column into a new 2 ml collection tube (included).
- Add 500 μl of Wash Buffer I (with ethanol added). Centrifuge for 1 min at 8000 x g. Discard the flow-through and place the purification column back into the collection tube.
- Add 500 μl of Wash Buffer II (with ethanol added) to the GeneJET™ Genomic DNA Purification Column. Centrifuge for 3 min at maximum speed (≥12000 x g). [Optional. If residual solution is seen in the purification column, empty the collection tube and re-spin the column for 1 min. at maximum speed. Discard the collection tube containing the flow-through solution and transfer the GeneJET™ Genomic DNA Purification Column to a sterile 1.5 ml microcentrifuge tube (not included).]
- Add 200 μl of Elution Buffer to the center of the GeneJET™ Genomic DNA Purification Column membrane to elute genomic DNA. Incubate for 2 min at room temperature and centrifuge for 1 min at 8000 x g.
- Discard the purification column. Use the purified DNA immediately in downstream applications or store at -20°C.

NOTES
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- For maximum DNA yield, repeat the elution step with additional 200 μl of Elution Buffer.
- If more concentrated DNA is required or DNA is isolated from a small amount of starting material.
- The volume of the Elution Buffer added to the column can be reduced to 50-100 μl. Please be aware that smaller volumes of Elution Buffer will result in smaller final quantity of eluted DNA.

TROUBLESHOOTING
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Low yield of purified DNA
- Excess sample used during lysate preparation.
Reduce the amount of starting material. Do not use more tissue or cells than indicated in lysis protocols.
- Starting material was not completely digested.
Extend the Proteinase K digestion at 56°C until complete lysis occurs and no particles remain.
- Ethanol was not added to the lysate.
Make sure that the ethanol was added to the lysate before applying the sample to the Purification Column.
- Ethanol was not mixed with the lysate.
After the addition of ethanol to the lysate mix the sample by vortexing or pipetting.
- Ethanol was not added to Wash Buffers.
Make sure that ethanol was added to Wash Buffer I and Wash Buffer II before use. Follow the instructions for Wash
Buffer preparation on p.3.

Purified DNA is degraded
- Sample was frozen and thawed repeatedly.
Avoid repeated freeze / thaw cycles of the samples. Use a new sample for DNA isolation. Perform extractions from fresh material when possible.
- Inappropriate sample storage conditions. Store bacteria at -20°C until use.

RNA contamination
- RNase A treatment was not carried out.
Carry out RNase A treatment step described in the purification procedure.

Column becomes clogged during purification
- Excess sample was used during lysate preparation.
Reduce the amount of starting material. A maximum of 2x10^9 of bacteria cells, 5x10^6 of suspension cells is recommended for lysate preparation.
- Tissue was not completely digested.
Extend the Proteinase K digestion at 56°C until complete lysis occurs and no particles remain.

Inhibition of downstream enzymatic reactions
- Purified DNA contains residual ethanol.
If residual solution is seen in the purification column after washing the column with Wash Buffer II, empty the collection tube and re-spin the column for an additional 1 min. at maximum speed (≥12000 x g).
- Purified DNA contains residual salt.
Use the correct order for the Washing Buffers. Always wash the purification column with Wash Buffer I first and then proceed to washing with Wash Buffer II.