Introduction of SARS‑CoV‑2 RNA Extraction using Biocomma Kit
Table of Contents
Biocomma nucleic acid (DNA/RNA) extraction kit is a Chinese product that is useful for RNA extraction systems for COVID-19 detection. It is applied for manual extraction and it is completed in four steps-
- Lysis
- Binding
- Washing and
- Elution.
A kit (for 50 tests) contains-
- Lysis buffer(GLX)-30 ml
- Proteinase K-20 mg/ml (1 ml)
- Spin column and collection tube(50/50 pieces)
- Wash buffer-1(PD)- 15 ml+15 ml isopropanol (outside)
- Wash buffer-2 ( PW)-15 ml+60 ml absolute ethanol(outside)
- RNase-free double distilled water – 10 ml (In stead of elution buffer)
Reagent preparation for SARS‑CoV‑2 RNA Extraction
1. Lysis buffer (GLX) is ready for use.
2. Add 15 ml Isopropanol to wash buffer-1(PD).
3. Similarly, add 60 ml isopropanol to wash buffer-2 ( PW).
4. Bring proteinase k from storage at -20C.
5. RNase-free ddH20-10 ml is for the elution step in place of the elution buffer.
Extraction Requirements
- Extraction kit
- Molecular Biology Grade Alcohol(Ethanol)
- Biological Safety Cabinet(BSC)-2
- Centrifuge
- Micropipettes and tips
- Waste containers
- Eppendorf tubes( 1.5 ml and 2.0 ml)
- Tissue paper rolls
- PPE sets
- Test specimen/nasopharyngeal swab/oropharyngeal swab
Extraction Procedure
1. Add 500 μL lysis buffer and 20 μL proteinase K in a centrifuge tube or Eppendorf tube for each test.
2. Now add 200μL sample.
4. Mix using a vortex for 1 minute and then incubate for 5-10 minutes.
6. Transfer the spin column (filter tube).
7. Centrifuge at 12000 rpm for 1 minute.
8. Discard filtrate.
9. Add 500 μL wash buffer-1 (PD).
10. Again centrifuge at 12000 rpm for 1 minute.
11. Discard filtrate.
12. Now put 700 μL wash buffer-2 (PW).
13. Centrifuge at 12000 rpm for 1 minute.
14. Discard filtrate.
15. Repeat steps 12,13 and 14.
16. Dry the spin column at 12000 rpm for 2 minutes.
17. Discard the tube (collection tube) from the spin column.
18. Keep the spin column/filter in a new centrifuge/ Eppendorf tube of 1.5 ml.
19. Add (RNase- free ddH20) 50- 100 μL slowly drop by drop at the Centre of the spin column so that it spreads easily over the column.
20. Finally centrifuge at 12000 rpm for 2 minutes.
21. Now throw the spin column and take the Eppendorf tube that has a template.
22. If delay in the master mix, maintain the cold chain.
Keynotes on SARS‑CoV‑2 RNA Extraction using Biocomma Kit
- This kit is useful for virus DNA/RNA Extraction (Spin Column).
- It can extract nucleic acid (DNA/RNA) from blood, tissue, organs, environmental sample, saliva, and nasal sample, and maximum remove impurities protein and other organic compound impurities.
- The extracted viral nucleic acid (DNA/RNA) can be directly used for downstream applications like PCR, RT-PCR, qPCR, and qRT-PCR experiment.
- The extraction procedure is simple, quick, and non-toxic with high-purity DNA/RNA and stable quality.
Bibliography
- https://www.biocomma.com/solutions/87.html
- https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7122149/