The following analysis of possible problems in Buccal swab/FTA card DNA extraction is helpful to your experiment. In addition, for other experimental or technical problems in addition to operating instructions and problem analysis, we have dedicated technical support to help you. If you have any needs, please contact us at: 028-83360257 or E-mali:Tech@foregene.com.

The purification column is clogged

In this kit, in the genomic DNA extraction operation, the purification column is directly adsorbed on the sample enzymatic lysis mixture without centrifugation step, and the purification column may be blocked due to incomplete enzymization and high viscosity of the sample.

The following possible causes are as follows:

1. Incomplete enzymatic digestion of tissue samples.

Recommendation: The sample processing time of Foregene Protease can be appropriately extended or the supernatant can be taken after centrifugation at 12,000 rpm (~13,400 × g) for 5 min.

2. Excessive use of tissue samples or large tissues.

Recommendation: It is best not to exceed 1 Buccal swab in the sample; if the sample is too large, increase the dosage of Buffer ST1, Foregene Protease, buffer ST2 accordingly.

3. The sample viscosity is too high.

Recommendation: Samples can be appropriately diluted with 10 mM of Tris-HCl before genomic DNA extraction.

4. Fragments of the blood card have been sucked.

Recommendation: The transient centrifugation time of step 6 of blood spot (FTA Card) genomic extraction can be appropriately extended.

Low yield or no DNA

There are often a variety of factors that affect genomic DNA yield, including sample origin, sample storage conditions, sample preparation, manipulation, etc.

Genomic DNA cannot be obtained during extraction

The possible causes are as follows:

1. Improper preservation of samples or storage for too long leads to genomic DNA degradation.

Recommendation: Oral swabs should preferably be freshly sampled, and it is not advisable to use preserved swabs for genomic DNA extraction operations; blood spot samples should ensure that the quality is qualified and the storage time should not be too long.

2. Too little tissue usage may result in no extraction of the corresponding genomic DNA.

Recommendation: Follow the buccal swab sampling instructions in the operation guide, and wipe as many times as possible so that enough cells can be attached to the oral swab for genomic DNA extraction; for blood spot sample extraction, the blood spot cutting area can be appropriately increased.

3. Foregene Protease is improperly preserved, resulting in a decrease in its activity or inactivation.

Recommendation: Confirm the storage conditions of the Foregene Protease or replace it with a new Foregene Protease for enzymatic reaction.

4. Improper preservation of the kit or storage time is too long, resulting in the failure of some components in the kit.

Recommendation: Purchase a new Buccal swab DNA isolation kit for related procedures.

5. Buffer WB does not add absolute ethanol.

Recommendation: Confirm that buffer WB adds the correct volume of absolute ethanol.

6. The eluent is not correctly added to the silicone film.

Recommendation: Add 65 °C pre-warmed eluent drops to the middle of the silicone membrane and leave at room temperature for 5 min to increase the elution efficiency.

Low-yield genomic DNA isolated

The following possible causes are as follows:

1. Improper preservation of samples or storage for too long leads to genomic DNA degradation.

Recommendation: Oral swabs are preferably freshly sampled, and preserved swabs should not be used for genomic DNA extraction.

2. If the amount of tissue sample is too small, the extracted genomic DNA content will be less.

Recommendation: Follow the oral swab sampling instructions in the operating guide, wiping as many times as possible so that enough cells can be attached to the oral swab for genomic DNA extraction.

3. Foregene Protease is improperly preserved, resulting in a decrease in its activity or inactivation.

Recommendation: Confirm the storage conditions of the Foregene Protease or replace it with a new Foregene Protease for enzymatic reaction.

4. Eluent problems.

Recommendation: Use Buffer EB for elution; if using ddH₂O or other eluents, confirm that the pH of the eluate is between 7.0-8.5.

5. The eluate is not correctly added dropwise.

Recommendation: Add eluent drops to the middle of the silicone membrane and leave at room temperature for 5 min to increase elution efficiency.

6. The elution liquid accumulates too little.

Recommendation: Use eluent for genomic DNA elution as required in the instructions, at least no less than 15 μl.

Low purity of genomic DNA isolated

Low genomic DNA purity can lead to failure or unsatisfactory results of downstream experiments, such as: enzymes cannot be cut open, PCR can not get the gene fragment of interest, etc.

The possible causes are as follows:

1. Heteroprotein pollution, RNA pollution.

Analysis: The purification column was not washed using Buffer PW; the Buffer PW wash purification column was not washed using the correct centrifugal speed.

Recommendation: Ensure that there is no precipitation in the supernatant before adding ethanol; be sure to wash the purification column according to the instructions, and this step cannot be omitted.

2. Impurity ion pollution.

Analysis: Buffer WB wash purification column was omitted or washed only once, resulting in residual ionic contamination.

Recommendation: Be sure to wash Buffer WB 2 times as directed to remove residual ions as much as possible.

3. RNA enzyme contamination.

Analysis: Foreign RNases were added to buffer; Buffer PW wash operation was incorrect, resulting in RNase residues, affecting downstream RNA experimental operations, such as in vitro transcription.

Recommendation: Foregene series nucleic acid isolation kits can remove RNA without additional addition of RNase,thus buccal Swab/FTA Card DNA Isolation Kit needn’t add RNase; be sure to follow the instructions for Buffer PW washing purification column, and this step cannot be omitted.

4. Ethanol residue.

Analysis: Buffer WB did not perform empty tube centrifugation after washing the purification column.

Recommendation: Perform the correct empty tube centrifugation operation according to the instructions.

5. Other impurity pollution.

Analysis: Saved samples or special samples are not pretreated.

Recommendation: Thoroughly pretreat the sample as instructed.