Revision as of 03:01, 6 May 2026

DNA extraction using HotShot

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:

place the specimen into 10–15 μl of alkaline buffer in a microvial
incubate the sample at 65°C for 18 mins + 98°C for 2 mins in a PCR machine
add the same amount of neutralizing buffer as you used of the alkaline buffer (so 10 μl alkaline + 10 μl neutralizing or 15 μl alkaline + 15 μl neutralizing)

@@ Line 23: / Line 23: @@
 === How to use the buffers to extract DNA ===
-* 10–15 μl of alkaline buffer + specimen
+# place the specimen into 10–15 μl of alkaline buffer in a microvial
-* Incubate at 65°C for 18 mins + 98°C for 2 mins
+# incubate the sample at 65°C for 18 mins + 98°C for 2 mins in a PCR machine
-* Add the same amount of neutralizing buffer
+# add the same amount of neutralizing buffer as you used of the alkaline buffer (so 10 μl alkaline + 10 μl neutralizing or 15 μl alkaline + 15 μl neutralizing)