Specimen Synthesis Lab Report
Specimen Preparation:
Samples and all test reagent and kit were brought into room temperature. In 2ml eppendrof tube approximately 220mg stool sample were taken. Washing buffer was prepared by adding distilled water .
Procedure for Purification of DNA from Stool sample:
i. 220mg stool sample were collected in a 2ml tubes and placed it on ice. ii. Added 2ml Buffer ASL to each stool tube. Used pipet to wash the stool sample from the spoon while transferring the buffer. Vortexed continuously for 1minute or until the stool samples was thoroughly homogenized. iii. Pipet 1.6ml of the stool lysate into a labeled 2 ml microcentrifuge tube. iv. Heated the suspension for 5 minutes at 70°C.
v. Then again vortexed for 15 seconds and centrifuge sample at full speed for …show more content…
1 monute to pellete stool particles. vi. Pipet 1.2 ml of the supernatant into a new 2ml microcentrifuge tube and discard the pellet. vii. Added 1 Inhibit buffer to each sample and vortex immediately and continuously for 1 minute. Incubated suspension for 1 minute at room temperature to allow inhibitors to adsorb to the Inhibitor matrix. viii. Centrifuge sample at full speed for 3 minutes to pellet stool particles. ix. Pipet all the supernatant into a new 1.5 ml microcentrifuge tube and discard the pellet. Centrifuged the sample at full speed for 3 minutes.
x. Pipet 15 µl proteinase K into a new 1.5 ml microcentrifuge tube. xi. Pipet 200 µl supernatant from step 9 into the 1.5 ml microcentrifuge tube containing proteinase K. xii. Added 200 µl Buffer AL and vortexed for 15 second.
DNA purification from Tissues:
• Excise the tissue sample or remove it from storage
• Cut up to 25 mg of tissue into small pieces.
Placed in a 1.5 ml microcentrifuge tube, and add 180 µl of Buffer ATL.
• Added 20 µl proteinase K, mixed by vortexing, and incubated at 56°C until the tissue is completely lysed. Vortex occasionally during incubation to disperse the sample.
• Added 200 µl Buffer AL to the sample, mixed by pulse-vortexting for 15 seconds and incubated at 70°C for 10 minutes. Briefly centrifuge the 1.5 ml microcentrifuge tube to remove drops from inside the lid.
• Added 200 µl ethanol to the sample and mix by pulse-vortexing for 15 seconds.
• Applied the mixture to the QIAamp Mini spin column and centrifuge at 6000×g (8000rpm) for 1 minute. Placed the QIAamp Mini spin column in a clean 2 ml collection tube.
• 500 µl Buffer AW1 were added and centrifuge at 6000× g (8000rpm) for 1 minute.
• Then 500 µl Buffer AW2 were added and centrifuge at full speed (20,000 × g; 14000rpm) for 4 minutes.
• Placed the QIAamp Mini spin column in a new 2 ml collection tube and centrifuge at full speed for 1 minute.
• Placed the Mini spin column in a new 1.5 ml microcentrifuge tube and added 50 µl Buffer AE and incubate at room temperature for 5 minutes and then centrifuge at 6000× g (8000 rpm) for 1
minute.
Samples and all test reagent and kit were brought into room temperature. In 2ml eppendrof tube approximately 220mg stool sample were taken. Washing buffer was prepared by adding distilled water .
Procedure for Purification of DNA from Stool sample:
i. 220mg stool sample were collected in a 2ml tubes and placed it on ice. ii. Added 2ml Buffer ASL to each stool tube. Used pipet to wash the stool sample from the spoon while transferring the buffer. Vortexed continuously for 1minute or until the stool samples was thoroughly homogenized. iii. Pipet 1.6ml of the stool lysate into a labeled 2 ml microcentrifuge tube. iv. Heated the suspension for 5 minutes at 70°C.
v. Then again vortexed for 15 seconds and centrifuge sample at full speed for …show more content…
1 monute to pellete stool particles. vi. Pipet 1.2 ml of the supernatant into a new 2ml microcentrifuge tube and discard the pellet. vii. Added 1 Inhibit buffer to each sample and vortex immediately and continuously for 1 minute. Incubated suspension for 1 minute at room temperature to allow inhibitors to adsorb to the Inhibitor matrix. viii. Centrifuge sample at full speed for 3 minutes to pellet stool particles. ix. Pipet all the supernatant into a new 1.5 ml microcentrifuge tube and discard the pellet. Centrifuged the sample at full speed for 3 minutes.
x. Pipet 15 µl proteinase K into a new 1.5 ml microcentrifuge tube. xi. Pipet 200 µl supernatant from step 9 into the 1.5 ml microcentrifuge tube containing proteinase K. xii. Added 200 µl Buffer AL and vortexed for 15 second.
DNA purification from Tissues:
• Excise the tissue sample or remove it from storage
• Cut up to 25 mg of tissue into small pieces.
Placed in a 1.5 ml microcentrifuge tube, and add 180 µl of Buffer ATL.
• Added 20 µl proteinase K, mixed by vortexing, and incubated at 56°C until the tissue is completely lysed. Vortex occasionally during incubation to disperse the sample.
• Added 200 µl Buffer AL to the sample, mixed by pulse-vortexting for 15 seconds and incubated at 70°C for 10 minutes. Briefly centrifuge the 1.5 ml microcentrifuge tube to remove drops from inside the lid.
• Added 200 µl ethanol to the sample and mix by pulse-vortexing for 15 seconds.
• Applied the mixture to the QIAamp Mini spin column and centrifuge at 6000×g (8000rpm) for 1 minute. Placed the QIAamp Mini spin column in a clean 2 ml collection tube.
• 500 µl Buffer AW1 were added and centrifuge at 6000× g (8000rpm) for 1 minute.
• Then 500 µl Buffer AW2 were added and centrifuge at full speed (20,000 × g; 14000rpm) for 4 minutes.
• Placed the QIAamp Mini spin column in a new 2 ml collection tube and centrifuge at full speed for 1 minute.
• Placed the Mini spin column in a new 1.5 ml microcentrifuge tube and added 50 µl Buffer AE and incubate at room temperature for 5 minutes and then centrifuge at 6000× g (8000 rpm) for 1
minute.