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Troubleshooting Guide:

DNA Extraction

PROBLEM:CAUSE:ANSWER:
There is no cell pellet after centrifuging the cheek cell suspension Not enough cheek cells in suspension. Mouth must be vigorously rinsed for at least 60 seconds to harvest loose cheek cells.
Sample not centrifuged fast enough. Spin cells at maximum speed (17,000 x g) for 2 minutes. If your centrifuge does not reach this speed, spin at highest available speed for 4 minutes.
I was not able to extract DNA from hair Not enough hairs used for extraction. Use at least five hairs for DNA extraction.
No follicle was present on hair shaft. The best place to collect hairs for this experiment is the head. Pick hair follicles which have a bulbous base (sheath ends).
Poor DNA Extraction Samples not mixed well enough during extraction. In addition to flicking the tube, vortex or pipet up and down to mix the sample.
Proteinase K inactive because it was prepared too far in advance. Prepare Proteinase K within one hour of use.
Water baths not at proper temperature. Use a thermometer to confirm water bath set point.
Not enough DNA Try cheek cell extraction. Final DNA concentrations are usually higher.
The extracted DNA is very cloudy Cellular debris from pellet transferred to tube. Centrifuge sample again and move supernatant to a fresh tube. Take care to avoid pellet.
Cellular debris not separated from supernatant. Centrifuge sample again. If possible, centrifuge at a higher speed. Move cleared supernatant to a fresh tube.

Human PCR

PROBLEM:CAUSE:ANSWER:
After staining, the ladder and control PCR products are visible on gel, but some student samples are not present Student DNA sample was not concentrated enough. Poor DNA extraction. Extract new DNA. Cheek cell extraction usually results in higher DNA yield.
Student DNA sample was degraded. If DNA is not used immeidately following extraction, store sample at -20˚C.
Wrong volumes of DNA and primer added to PCR reaction. Practice using pipettes.
Some students have more or less amplification than others Concentration of DNA varies by sample. There is an inherent variability in the extraction process. For best results, use cheek cell extraction.
Low molecular weight band in PCR samples Primer dimer Low concentration of extracted DNA in PCR reaction.

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