Okay, I found a few lab services that do this for cheap. Your protocol is well written out so I don't think I'll have any trouble. All I need is the primers used and I think I'll be set. Thanks for the help. 

On Wednesday, December 19, 2012 2:00:02 AM UTC-5, Nathan McCorkle wrote:

mt-23F and mt-23R

see the protocol I followed here:

http://nathanmccorkle.com/pdf/RIT_stuff/Project%205%20Protocol-2008.pdf

On Tue, Dec 18, 2012 at 10:37 PM, kishb87 <kis...@gmail.com> wrote:

Hi Nathan, 

Thanks for the help! Do you remember which primers you used for HVR-1 sequencing? Also, I'm not quite following the clean PCR step. And what exactly did the lab professional do to evaluate the sample? 

Kishan

On Tuesday, December 18, 2012 6:45:06 PM UTC-5, Nathan McCorkle wrote:

I wrote this up when we did it in an Intro Bio lab course... I can't remember if I have a final copy somewhere, but it looks mostly complete

---------- Forwarded message ----------
From: Nathan McCorkle <nmz...@gmail.com>
Date: Mon, Feb 23, 2009 at 5:03 PM
Subject: Re: lab
To: old lab mates

I stayed too late and didnt give my laptop to anyone.... here is the paper, I finished up to the end of electrophoresis

Map of process:

Obtain cells.

Extract DNA

Measure and evaluate sample quality

Replicate target sequence with PCR

Visualize sample with gel electrophoresis

Clean sample

Perform sequencing PCR

Evaluate sequenced sample (have a professional lab do this)

Evaluate sequence data using Bio-Informatics software

Compare resulting information to others using internet databse

Be sure to inform students that they should not eat, drink, or brush their teeth for at least 1 hour before the laboratory session begins.

Buccal (cheek) cells are obtained by vigorously rinsing one's mouth with ethyl alcohol (EtOH) based Scope mouthwash and spitting into a centrifuge test tube. In this step the chemical properties of ethanol (EtOH) are important because it weakens interactions between cells allowing them to separate and go into suspension, and is aided by the mechanical stress provided the rinsing. It is important to thoroughly perform this step in order to obtain a sufficient quantity of cells to extract DNA from. The sample is centrifuged to collect cells in a pellet at the bottom of the centrifuge tube, removing them from the liquid Scope, which is then poured off. The rotation of the samples in the centrifuge induces centripetal force on the contents of the tubes which separates compounds based on their mass in relation to rotational speed and distance from axis of rotation.

The pellet is resuspended in cell lysis solution which includes a mild non-ionic detergent, Triton-X, and a pH buffer. The detergent interacts with the cellular membranes, dissolving them and allowing cellular contents to disperse into solution. This solution should be mixed well and allowed to sit at room temperature for 15 minutes.

Proteins in the sample may interfere and inhibit a proper PCR reaction, or could even be harmful nuclease enzymes that destroy DNA. To denature these proteins, Proteinase K solution is then added to the sample, mixed well, and allowed to incubate at room temperature for 10 minutes. Proteinase K is an enzyme that cleaves the peptide bonds between individual amino acids which form a protein. To separate DNA from denatured protein, protein precipitation solution is added to the sample, mixed, and allowed to incubate on ice for 10 minutes. This solution contains the salt ammonium sulfate, which when dissolved in aqueous solution has a higher affinity than amino acids to water. This eventually decreases water–amino acid interaction so much that protein subunits fall out of solution, a process known as salting-out. This solution is then centrifuged, a protein pellet forms at the bottom of the tube while the DNA remains dissolved in solution. The liquid is transferred to and mixed with a tube containing isopropanol and glycogen solution and centrifuged for 5 minutes. The protein pellet is discarded, and the DNA containing solution is centrifuged. Glycogen posses' a net like structure which entangles DNA acting as a carrier. Both are insoluble in alcohols, causing the two to precipitate together. After centrifugation completes, the isopropanol is poured off and ethanol is added to wash the pellet. After gently inverting the tube several times, centrifuge again for 1 minute and pour off ethanol. Invert tube and blot rim with absorbent paper towel to wick away any remaining alcohol, and resuspend in DNA Hydration solution, and incubate at 65^o C for 1 hour, and store at 4^o C. The DNA hydration solution contains a pH buffer and a mineral chelator EDTA. EDTA binds minerals that some enzymes need to perform their function, preventing harmful enzymes from degrading our DNA.

Quantify the amount of DNA and protein in the sample by using a NanoDrop photospectrometer. The NanoDrop is designed specifically to require only a small amount of sample, minimizing the loss of DNA isolate. Photospectrometry measures the amount of light a sample absorbs, with optimal wavelengths for DNA and protein in the UV range at 260nm and 280nm, respectively. Using the Beer-Lambert equation, concentrations of DNA and protein within in a sample can be calculated, though these calculations are performed by the NanoDrop software for ease. The optimal ratio of DNA to protein is >1.8 with allowances as far down as 1.6, if the DNA:protein ratio is less than 1.6, the sample is too contaminated, and if there is greater than 150ng/ul DNA the sample must be diluted.

The previously isolated buccal cell DNA will be used as a template to isolate and amplify the mitochondrial genome's hypervariable region (HVR) sequence using polymerase chain reaction (PCR). PCR utilizes DNA polymerase synthesizes DNA by adding free high-energy DNA bases (dNTPs) complementary to a template strand in a 3' – 5' direction, from a starting point known as a primer. Primers are short sequences of about 20 bases which are complementary to areas flanking the sequence of interest, the forward primer to anneal near the beginning of sequence on the positive sense strand, and the reverse primer to anneal near the end of the sequence with the negative sense strand.

These primers are mixed with activated DNA bases (dNTPs), Taq polymerase (heat stable polymerase), and a pH buffer containing MgCl₂ (Mg is an important divalent ion needed for polymerase function), together known as a PCR master mix. Appropriate proportions of the master mix and template DNA are combined; the mixture is heated to 95^oC melting and separating the DNA helix into two strands; cooled to 62^oC to allow primers to anneal to their complements; heated to 72^oC until Taq polymerase completes extension from the primer, resulting in new strands. The process of melting, annealing, and extension is repeated approximately 30 times, with new strands serving as templates in each subsequent cycle.

PCR is known as a chain reaction because each cycle doubles the amount of template DNA to be used in the next cycle. Strands synthesized using the original template are characterized by beginning with a primer but extending past the end of the target sequence, however and strands synthesized from this second-generation strand will begin in the extended region, and terminate where the first primer began. This means that some strands will actually be longer than the target sequence, but because PCR proceeds commonly for at least 30 cycles, of the many billions of resulting copies, statistically few of them will have this overhang present.

To confirm success of the PCR reaction, gel electrophoresis is used to separate DNA fragments by sequence length, allowing a comparison of expected PCR product to actual PCR product. Gel electrophoresis uses electrical current to pull DNA strands through a gel made of agarose polysaccharides. The agarose is dissolved in a conductive buffer and allowed to solidify in a square mold with a plastic comb placed near an edge and hanging so as to create depressions or wells in the gel which. Once solidified, the comb is remove and the gel is placed into a rectangular tray with electrodes at either end, submerged in the same conductive buffer, and DNA samples loaded into the wells with loading dye that allows a user to track migration as power is applied, to prevent samples from running out the end of the gel. Agarose forms a porous matrix, and the negatively charged DNA is pulled towards the positive electrode as the electrical current flows through the gel. The speed that DNA travels through the gel is relative to the number of nucleotide bases, concentration of agarose, and amount of electrical power. By keeping agarose gel concentration and electrical power constant, speed of DNA migration through the gel will vary according to its size alone. Small fragments move faster than longer strands because they slip through the pores of the matrix more easily than larger strands, creating a gradient of fragment sizes. To determine DNA length the sample must be compared to a controlled standard known as a DNA ladder. DNA ladders are comprised of a mixture of synthetic DNA segments of known length, with a range relative to the sample to be compared. Dyes which specifically stain DNA can be dissolved in the gel or applied later after migration is complete, either way provides similar results, DNA can be viewed by shining UV light on the stained gel, appearing as bands along the path of current flow.

Clean the PCR sample by cleaving the single-stranded nucleotide "loose ends," degrading the unused dNTP, and cleaving 5' phosphate groups to prevent ligation. Exonuclease degrades single strands starting at 3', and alkaline phosphatase catalyzes the removal of phosphate groups from both 5' and 3' ends, and from single nucleotides. The absence of 5' phosphate groups allows the addition of radioactive phosphates there which fluoresce in the DNA sequencer.

Perform 25 thermal cycles of PCR using a solution of Sequencing Master Mix and cleaned PCR sample. With this solution, the PCR reaction randomly terminates the addition of nucleotides with fluorescently labeled ddNTPs (dNTP lacking 3' -OH group) allowing a range of sequence lengths to be produced with high statistical probability of every sub -length being produced, from minimum (primer +1) to maximum (complete original sequence.)

Send the sequenced PCR sample to a lab for reading.

Compare returned sample reading to Cambridge standard, record differences.

Search Mitosearch internet database using sample differences, evaluate results and note ethnicity and country of origin.

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