Methods/DNA barcoding: Difference between revisions
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Created page with " = DNA EXTRACTION USING HOTSHOT = == MIXING NEW BUFFER == === Preparation === '''Alkaline Buffer''' (= 25 mM NaOH + 0.2 mM disodium EDTA) # Premix 10M NaOH: #* 70 ml H₂O + 40g NaOH and adjust to 100 ml #* ''or'' #* 17.5 ml H₂O + 10g NaOH and adjust to 25 ml # To 25 ml of H₂O add: #* 62.5 μl of 10M NaOH #* 10 μl of 0.5 M disodium EDTA ---- '''Neutralizing Buffer''' (40 mM trisHCl) Mix the following: * 24 ml of H₂O * 1 ml of 1M trisHCl ---- === Extracti..." |
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= DNA | = DNA extraction using HotShot = | ||
== | === Mixing the buffers === | ||
'''Alkaline Buffer''' (= 25 mM NaOH + 0.2 mM disodium EDTA) | '''Alkaline Buffer''' (= 25 mM NaOH + 0.2 mM disodium EDTA) | ||
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#* 62.5 μl of 10M NaOH | #* 62.5 μl of 10M NaOH | ||
#* 10 μl of 0.5 M disodium EDTA | #* 10 μl of 0.5 M disodium EDTA | ||
'''Neutralizing Buffer''' (40 mM trisHCl) | '''Neutralizing Buffer''' (40 mM trisHCl) | ||
Revision as of 02:45, 6 May 2026
DNA extraction using HotShot
Mixing the buffers
Alkaline Buffer (= 25 mM NaOH + 0.2 mM disodium EDTA)
- Premix 10M NaOH:
- 70 ml H₂O + 40g NaOH and adjust to 100 ml
- or
- 17.5 ml H₂O + 10g NaOH and adjust to 25 ml
- To 25 ml of H₂O add:
- 62.5 μl of 10M NaOH
- 10 μl of 0.5 M disodium EDTA
Neutralizing Buffer (40 mM trisHCl)
Mix the following:
- 24 ml of H₂O
- 1 ml of 1M trisHCl
Extraction
- 10–15 μl of alkaline buffer + specimen
- Incubate at 65°C for 18 mins + 98°C for 2 mins
- Add the same amount of neutralizing buffer
