Jul 11, 2022
Extraction of DNA from endocervical samples using QIAamp DNA mini kit
- emmanuelgomseu EBDG Boris Emmanuel DJOUMSIE GOMSEU1,2,3,4,5
- 11Department of Internal Medicine, Hallym University Hangang Sacred Heart Hospital, Seoul 07247, Korea;
- 22Department of Environmental Biology and Medical Parasitology;
- 33Department of Biomedical Science, Hanyang University Graduate School of Biomedical Science and Engineering;
- 44Department of Urology, Hanyang University College of Medicine, Seoul 04763, Korea;
- 55Department of Parasitology and Tropical Medicine, Kyungpook National University School of Medicine, Daegu 41944, Korea
- Emma34
Document Citation: emmanuelgomseu EBDG Boris Emmanuel DJOUMSIE GOMSEU 2022. Extraction of DNA from endocervical samples using QIAamp DNA mini kit. protocols.io https://dx.doi.org/10.17504/protocols.io.btnvnme6
License: This is an open access document distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Created: March 26, 2021
Last Modified: July 11, 2022
Document Integer ID: 48565
Keywords: using qiaamp dna mini kit, qiaamp dna mini kit, extraction of dna, discard the qiaamp mini spin column, placed qiaamp mini spin column, qiaamp mini spin column, qiaamp mini kit, endocervical sample, incubate at room temperature, endocervical specimen, centrifuge tube, centrifuge the spin column, times during incubation, extraction, subsequent molecular analysis, incubation, dna, homogenized mixture
Abstract
DNA was extracted from endocervical samples using the QIAamp mini kit according to the manufacturer’s instructions.
1 Centrifuge tubes containing the endocervical specimens at 15,000 rpm for 00:10:00 .
2 Remove supernatant and preserve the pellet;
3 Add 180 µL of the ATL buffer and homogenize with the help of a vortex mixer.
4 Add 20 µL Proteinase K to the homogenized mixture and mixe by vortexing
5 Incubate the solution in a dry bath at 56 °C for 03:00:00 while homogenizing 2 to 3 times during incubation.
6 Add 200 μL AL of buffer and homogenize again using a vortex for 00:00:15
7 Incubate the solution in a dry bath at70 °C for 00:10:00 .
8 Add 200 µL of ethanol and homogenize.
9 Then transfer the mixture carefully into a QIAamp mini spin column and centrifuge at 8,000 rpm for 00:01:00 .
10 Discard filtrate and place the minicolumns were placed QIAamp mini spin column in new 2 mL tubes
11 Add of 500 µL buffer AW1, centrifuged at 8,000 rpm for 00:01:00 and discard the filtrate.
12 Add of 500 µL buffer AW2 was added to minicolumns.
13 Centrifuge at 14,000 rpm for 3 minutes and discard the filtrate.
14 Centrifuge the spin column with the collection tube again at 14,000rpm for 00:01:00
15 Place minicolumns in 1.5 mL micro-centrifuge tubes and add 50 µL of buffer AE.
16 Incubate at room temperature for 5 minutes and centrifuge the mixture at 10,000 rpm for 00:03:00 .
17 Discard the QIAamp mini spin column and store micro-centrifuge at -20 °C for subsequent molecular analysis.
Troubleshooting
DNA was extracted from endocervical samples using the QIAamp mini kit according to the manufacturer’s instructions.
1 Centrifuge tubes containing the endocervical specimens at 15,000 rpm for 10 minutes.
2 Remove supernatant and preserve the pellet;
3 Add 180 μL of the ATL buffer and homogenize with the help of a vortex mixer.
4 Add Proteinase K to the homogenized mixture and mixe by vortexing
5 Incubate the solution in a dry bath at 56 oC for 3 hours while homogenizing 2 to 3 times during incubation.
6 Add 200 μL AL of buffer and homogenize again using a vortex for 15 seconds
7 Incubate the solution in a dry bath at 70°C for 10 minutes.
8 Add 200 μL of ethanol and homogenize.
9 Then transfer the mixture carefully into a QIAamp mini spin column and centrifuge at 8,000 rpm for one minute.
10 Discard filtrate and place the minicolumns were placed QIAamp mini spin column in new 2 mL tubes
11 Add of 500 μL buffer AW1, centrifuged at 8,000 rpm for one minute and discard the filtrate.
12 Add of 500 μL buffer AW2 was added to minicolumns.
13 Centrifuge at 14,000 rpm for 3 minutes and discard the filtrate.
14 Centrifuge the spin column with the collection tube again at 14,000rpm for 1 minute
15 Place minicolumns in 1.5 mL micro-centrifuge tubes and add 50 μL of buffer AE.
16 Incubate at room temperature for 5 minutes and centrifuge the mixture at 10,000 rpm for 3 minutes.
17 Discard the QIAamp mini spin column and store micro-centrifuge at -20 °C for subsequent molecular analysis.