Methods/DNA barcoding - Revision history

Admin at 01:09, 6 May 2026

2026-05-06T01:09:57Z

← Older revision		Revision as of 03:09, 6 May 2026
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	== Our extraction protocol ==		== Our extraction protocol ==

	~~'''BEFORE~~ starting~~'''~~, check if we have:		=== Before starting, check if we have:===
	* enough buffers (alkaline and neutralizing) – if not, mix new one		* enough buffers (alkaline and neutralizing) – if not, mix new one
	* enough sterile ~~stripes~~ – if not, autoclave more		* enough sterile strips – if not, autoclave more

	=== Prepare the Extraction Plate Map (MS Excel) ===		=== Prepare the Extraction Plate Map (MS Excel) ===
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	* one soft and two hard forceps (sterilize properly in the beginning)		* one soft and two hard forceps (sterilize properly in the beginning)

	=== Prepare ~~stripes~~ ===		=== Prepare strips ===
	# Prepare 12 sterile strips.		# Prepare 12 sterile strips.
	# Pipette '''15 μl of alkaline buffer into each microvial''' (use tips with filter)		# Pipette '''15 μl of alkaline buffer into each microvial''' (use tips with filter)
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	* Take one '''specimen from each sample''', put in 48-well plate in purified water		* Take one '''specimen from each sample''', put in 48-well plate in purified water
	* In '''large specimens''', take 1–3 legs (ideally each from one side) using hard forceps. Return the specimen to the original vial, and mark the vial label in red		* In '''large specimens''', take 1–3 legs (ideally each from one side) using hard forceps. Return the specimen to the original vial, and mark the vial label in red
	* After some time (~~5–15~~ mins) move the specimen from the water to the strip microvial with buffer using sterile forceps.		* After some time (c. 5 mins) move the specimen from the water to the strip microvial with buffer using sterile forceps.

	=== DNA extraction ===		=== DNA extraction ===
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	=== After DNA extraction ===		=== After DNA extraction ===
	* ~~'''~~Pipette out the liquid~~'''~~ into new marked strips. This will be your '''DNA extract'''		* Pipette out the liquid into new marked strips. This will be your '''DNA extract'''
	* ~~'''~~Add 75% or 95% alcohol~~'''~~ to the strips with specimens		* Add 75% or 95% alcohol to the strips with specimens
	* ~~'''~~Add Sample ID labels to each sample.~~'''~~		* Add Sample ID labels to each sample.

	=== Storing the extraction ===		=== Storing the extraction ===

Admin at 01:05, 6 May 2026

2026-05-06T01:05:57Z

← Older revision		Revision as of 03:05, 6 May 2026
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	# Place the specimen into 10–15 μl of alkaline buffer in a microvial		# 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		# 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)		# 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)

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	* select the samples you will extract		* select the samples you will extract
	* make a plate map, '''keep at least 2 positions empty'''		* make a plate map, '''keep at least 2 positions empty'''
	* save the plate map, and print it out
	* mark positions with BIG animals (they have "yes" in the sample sorting file)		* mark positions with BIG animals (they have "yes" in the sample sorting file)
	* upload the plate map into the Lab Cloud		* upload the plate map into the Lab Cloud and print it out twice (once to backup in the folder, the other copy will be used as labels after extraction)

	=== Prepare the place for work ===		=== Prepare the place for work ===

Admin at 01:03, 6 May 2026

2026-05-06T01:03:43Z

← Older revision		Revision as of 03:03, 6 May 2026
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	# Incubate the sample at 65°C for 18 mins + 98°C for 2 mins in a PCR machine		# 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)		# 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)


			== Our extraction protocol ==

			'''BEFORE starting''', check if we have:
			* enough buffers (alkaline and neutralizing) – if not, mix new one
			* enough sterile stripes – if not, autoclave more

			=== Prepare the Extraction Plate Map (MS Excel) ===
			* select the samples you will extract
			* make a plate map, '''keep at least 2 positions empty'''
			* save the plate map, and print it out
			* mark positions with BIG animals (they have "yes" in the sample sorting file)
			* upload the plate map into the Lab Cloud

			=== Prepare the place for work ===
			* 2× 48-well plastic tray, fill each well with a bit of purified water
			* burner with enough alcohol
			* lighter
			* sterile Petri dish with a bit of purified water
			* paper tissues
			* one soft and two hard forceps (sterilize properly in the beginning)

			=== Prepare stripes ===
			# Prepare 12 sterile strips.
			# Pipette '''15 μl of alkaline buffer into each microvial''' (use tips with filter)
			# Close all strips.
			# '''Number the strips''' – numbers 1–12 on the lid of the first microvial in the strip
			# Mark the empty positions.

			=== Put specimens into the alkaline buffer ===
			* Take one '''specimen from each sample''', put in 48-well plate in purified water
			* In '''large specimens''', take 1–3 legs (ideally each from one side) using hard forceps. Return the specimen to the original vial, and mark the vial label in red
			* After some time (5–15 mins) move the specimen from the water to the strip microvial with buffer using sterile forceps.

			=== DNA extraction ===
			# '''Put into PCR machine''' – the program is called '''HOTSHOT''' (65°C for 18 mins + 98°C for 2 mins)
			# Add '''15 μl of neutralizing buffer into each microvial'''.

			=== After DNA extraction ===
			* '''Pipette out the liquid''' into new marked strips. This will be your '''DNA extract'''
			* '''Add 75% or 95% alcohol''' to the strips with specimens
			* '''Add Sample ID labels to each sample.'''

			=== Storing the extraction ===
			* Double-check that the plate is properly marked (e.g., '''Extraction: Eric001''')
			* You can use it immediately for PCR
			* '''If you will use it''' soon (in 1–2 days), keep it in the '''-20°C freezer'''
			* '''If you will not use it''' soon or it is finalized, keep it in the '''-70°C freezer'''

Admin at 01:01, 6 May 2026

2026-05-06T01:01:30Z

← Older revision		Revision as of 03:01, 6 May 2026
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	=== How to use the buffers to extract DNA ===		=== How to use the buffers to extract DNA ===

	# ~~place~~ the specimen into 10–15 μl of alkaline buffer in a microvial		# 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		# 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)		# 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)

Admin at 01:01, 6 May 2026

2026-05-06T01:01:01Z

← Older revision		Revision as of 03:01, 6 May 2026
Line 23:		Line 23:
	=== How to use the buffers to extract DNA ===		=== 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)

Admin at 00:58, 6 May 2026

2026-05-06T00:58:41Z

← Older revision		Revision as of 02:58, 6 May 2026
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			=== How to use the buffers to extract DNA ===
	=== ~~Extraction~~ ===

	* 10–15 μl of alkaline buffer + specimen		* 10–15 μl of alkaline buffer + specimen
	* Incubate at 65°C for 18 mins + 98°C for 2 mins		* Incubate at 65°C for 18 mins + 98°C for 2 mins
	* Add the same amount of neutralizing buffer		* Add the same amount of neutralizing buffer

Admin at 00:58, 6 May 2026

2026-05-06T00:58:02Z

← Older revision		Revision as of 02:58, 6 May 2026
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	= DNA extraction using HotShot =		= DNA extraction using HotShot =

	=== Mixing the buffers ===		=== Mixing the buffers ===



	'''Alkaline Buffer''' (= 25 mM NaOH + 0.2 mM disodium EDTA)		'''Alkaline Buffer''' (= 25 mM NaOH + 0.2 mM disodium EDTA)

Admin at 00:57, 6 May 2026

2026-05-06T00:57:47Z

← Older revision		Revision as of 02:57, 6 May 2026
Line 4:		Line 4:

	=== Mixing the buffers ===		=== Mixing the buffers ===

Admin at 00:55, 6 May 2026

2026-05-06T00:55:30Z

← Older revision		Revision as of 02:55, 6 May 2026
Line 4:		Line 4:

	=== Mixing the buffers ===		=== 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)

Admin at 00:54, 6 May 2026

2026-05-06T00:54:49Z

← Older revision		Revision as of 02:54, 6 May 2026
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	* 24 ml of H₂O		* 24 ml of H₂O
	* 1 ml of 1M trisHCl		* 1 ml of 1M trisHCl



	=== Extraction ===		=== Extraction ===