DNA Extraction
A three millimeter square piece sample of unknown thawed tissue was selected using sterilized equipment. The A17 sample was placed in a steril in a 1.5mL Eppendorf tube. The cell membrane of the unknown meat was then broken down and the DNA was extracted by using the animal tissue extraction kit, Qiagen #69506. The final Eppendorf tube contained the DNA from A17 and was stored at -20 oC until gel electrophoresis.
Gel Electrophoresis
To determine the success of DNA extraction and later PCR clean-up the samples went through gel electrophoresis. DNA extraction was tested by putting the samples in the loading chambers of the supporting matrix which was made of one percent agarose gel. GelRed, a nucleic acid dye, was added to help visually identify the DNA bands. A power supply was added to the gel for 20 minute by placing an anode (positive poled) at the bottom and a cathode (negative poled) at the top. PCR clean-up was tested using the same electrophoresis method as the DNA extraction.
PCR
A portion of DNA from the extraction was amplified using polymerase chain reaction (PCR). A new sterile tube was used. In the tube 23.0µL of master mix was added which contained 10.5µL of water, 12.5µL Green Taq( premix with buffer, Taq, and dNTPs). Next 0.5µL …show more content…
For the sequencing reaction protocol two new Eppendorf tubes were used one was labeled ND4 and the other Leu. Using a new pipette, two microliters of stock PCR product were added to each tube. Then 5.5µL of ddH2O was added to each tube for a total 7.5µL of mix in each tube. Next, 0.5µL ND4 primer was added to the tube labeled ND4 and 0.5µL Leu primer was added to the tube labeled Leu. Two microliters of Big Dye Terminator Cycle Sequencing Buffer (mixture of ddNTPs, dNTPs, Taq polymerase, and salts) were added to each tube. The tubes were then capped and stored at twenty degrees Celsius until thermal cycling was
A three millimeter square piece sample of unknown thawed tissue was selected using sterilized equipment. The A17 sample was placed in a steril in a 1.5mL Eppendorf tube. The cell membrane of the unknown meat was then broken down and the DNA was extracted by using the animal tissue extraction kit, Qiagen #69506. The final Eppendorf tube contained the DNA from A17 and was stored at -20 oC until gel electrophoresis.
Gel Electrophoresis
To determine the success of DNA extraction and later PCR clean-up the samples went through gel electrophoresis. DNA extraction was tested by putting the samples in the loading chambers of the supporting matrix which was made of one percent agarose gel. GelRed, a nucleic acid dye, was added to help visually identify the DNA bands. A power supply was added to the gel for 20 minute by placing an anode (positive poled) at the bottom and a cathode (negative poled) at the top. PCR clean-up was tested using the same electrophoresis method as the DNA extraction.
PCR
A portion of DNA from the extraction was amplified using polymerase chain reaction (PCR). A new sterile tube was used. In the tube 23.0µL of master mix was added which contained 10.5µL of water, 12.5µL Green Taq( premix with buffer, Taq, and dNTPs). Next 0.5µL …show more content…
For the sequencing reaction protocol two new Eppendorf tubes were used one was labeled ND4 and the other Leu. Using a new pipette, two microliters of stock PCR product were added to each tube. Then 5.5µL of ddH2O was added to each tube for a total 7.5µL of mix in each tube. Next, 0.5µL ND4 primer was added to the tube labeled ND4 and 0.5µL Leu primer was added to the tube labeled Leu. Two microliters of Big Dye Terminator Cycle Sequencing Buffer (mixture of ddNTPs, dNTPs, Taq polymerase, and salts) were added to each tube. The tubes were then capped and stored at twenty degrees Celsius until thermal cycling was