Editing Saliva Preservation Buffer (section)

== Genomic DNA Isolation ==

DNA Stabilization Buffer (250 ml)

{| border="1" cellspacing="1" cellpadding="1" style="width:500px;"
|-
| <span style="line-height: 20.8px;">Component &nbsp;&nbsp;</span><br/>
| <span style="line-height: 20.8px;">Volume (ml)&nbsp;</span><br/>
| <span style="line-height: 20.8px;">[Final]</span><br/>
|-
| <span style="line-height: 20.8px;">1 M NaCl&nbsp;</span><br/>
| <span style="line-height: 20.8px;">1.461 g&nbsp;</span><br/>
| <span style="line-height: 20.8px;">0.1 M</span><br/>
|-
| <span style="line-height: 20.8px;">Tris HCl</span><br/>
| <span style="line-height: 20.8px;">2.5</span><br/>
| <span style="line-height: 20.8px;">0.01 M</span><br/>
|-
| <span style="line-height: 20.8px;">0.5M&nbsp;</span><span style="line-height: 20.8px;">EDTA</span><br/>
| <span style="line-height: 20.8px;">5</span><br/>
| <span style="line-height: 20.8px;">0.01 M</span><br/>
|-
| <span style="line-height: 20.8px;">10% SDS</span><br/>
| <span style="line-height: 20.8px;">12.5&nbsp;</span><br/>
| <span style="line-height: 20.8px;">0.014 M</span><br/>
|-
| <span style="line-height: 20.8px;">Proteinase K Solution (20 mg/ml)&nbsp;</span><br/>
| <span style="line-height: 20.8px;">2.5&nbsp;</span><br/>
| <span style="line-height: 20.8px;">&nbsp;6.92x10-6 M</span><br/>
|-
| <span style="line-height: 20.8px;">ddH2O</span><br/>
| <span style="line-height: 20.8px;">227.5 &nbsp;</span><br/>
| <br/>
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| <br/>
| <br/>
| <br/>
|-
| <br/>
| <br/>
| <br/>
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1. Saliva Collection and Storage

Prior to saliva collection, ensure that the subject’s mouth is free of food or other foreign substances by having the subject rinse their mouth with water and avoiding eating or drinking for 30 min before collecting the sample.

Open a 15 ml centrifuge tube with '''2.5 ml''' of DNA stabilization buffer&nbsp;making sure to avoid touching the inside of the cap or tube, and have the subject spit '''2.5 ml '''of saliva into the buffer solution.

Note: Collecting more than 2.5 ml of saliva can lead to sample degradation from an insufficient ratio of sample to DNA stabilization buffer. Collecting too little saliva will reduce expected yields from the protocol. To evaluate collection volumes, use the numbered gradients on the side of the tube.

Replace cap and mix by inversion until the mixture is homogenized. Vigorous shaking is not necessary.'''Store the samples at RT for short-term storage or 4 °C for long-term storage (>3 months).'''

2. Initial Preparations and Cell Lysis

Prior to starting the extraction, heat a water bath to 37 °C, and prepare an ice bucket. Three 15 ml conical centrifuge tubes will be needed for each extracted sample. The three tubes will be used to hold the cell and protein pellet, the final extracted gDNA, and the isopropanol and ethanol supernatants.

Retrieve samples from storage, and invert samples several times then vortex at medium speed for 15 sec.

Dispense 2.5 ml of sample into a clean 15 ml centrifuge tube, and add 5 ml of Cell Lysis Solution. Mix the sample 50 times by inversion, and incubate at RT for 30 min.

3. RNA Removal

Add '''40 μl of RNase A Solution at 100 mg/ml, and incubate at 37 °C for 15 min.'''

Remove the sample from the 37 °C water bath and cool on ice for 3 min.

After the RNase A incubation, increase the temperature of the water bath to 65 °C for the DNA rehydration step of the protocol.

4. Protein and Lipid Removal

Add 50 μl of Proteinase K Solution at 20 mg/ml, mix several times by inversion, and incubate at RT for a minimum of 30 min. Note: This is a possible pausing point for the protocol. After the addition of the Proteinase K Solution, the sample can be stored at 4 °C until the extraction can be completed. Storage at 4 °C for up to 24 hr was not shown to have a significant effect on the extraction yields or DNA quality. Long-term storage at this stage has not been evaluated.

Add 1.7 ml of Protein Precipitation Solution, vortex vigorously for 20 sec at high speed, and place on ice for 10 min.

Once the samples have cooled on ice for 10 min, centrifuge for 10 min at 3,000 x g and 4 °C. The precipitated proteins must form a tight pellet to continue. If the pellet is not tight or the solution is still cloudy, the samples can be cooled on ice for 5 min more and centrifugation repeated. The samples must be kept on ice to ensure a tight pellet.

5. Isolation and Purification of gDNA

Into a clean 15 ml centrifuge tube, pipet '''5 ml of Isopropanol '''and'''8 μl of pure Glycogen Solution at 20 mg/ml.'''

Pour the supernatant containing the gDNA from step 4.3 into the tube containing the Isopropanol and Glycogen Solution, leaving behind the precipitated protein pellet. Once the supernatant has been added, gently mix the sample 50 times by inversion and centrifuge for 30 min at 3,000 x g and 4 °C.

Pour the supernatant slowly into a clean 15 ml tube. After removal of the supernatant, add 1 ml of 70% ethanol to wash the pellet by slowly rocking and gently moving the ethanol over the precipitated pellet several times. Retain the ethanol in the tube.

After the initial wash, centrifuge the sample for 1 min at 2,000 x g and 20 °C. This centrifugation step can be done at either 4 °C or 20 °C. No significant effect of temperature has been shown for this step.

Following the initial wash and centrifugation of the pellet, slowly pour the ethanol wash from the tube and discard, then perform a second wash by repeating steps 5.3 and 5.4.

After the removal of the supernatant from the second wash, allow the pellet to air dry for 15 min.

If the sample has not completely dried, air dry for another 15 min.

6. Rehydration of gDNA

Once the sample has dried, add 300 μl of Tris-EDTA to rehydrate the dried gDNA pellet.

Vortex the sample for 5 sec at medium speed and place in a 65 °C hot water bath for 1 hr.

Remove the samples from the water bath and incubate O/N at RT.

Ref [https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4442313/ https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4442313/]