LongAmp PCR NEB - Revision history

Anbuser: /* Thermocycling Conditions */

2016-06-14T20:09:01Z

‎Thermocycling Conditions

Anbuser: /* Modifications: */

2016-06-14T19:53:51Z

‎Modifications:

Anbuser: /* Reaction Setup: */

2016-06-14T19:53:38Z

‎Reaction Setup:

Anbuser: /* Reaction Setup: */

2016-06-14T19:53:12Z

‎Reaction Setup:

Anbuser: /* Reaction Setup: */

2016-06-14T19:53:01Z

‎Reaction Setup:

Anbuser: /* Modifications: */

2016-06-14T19:52:13Z

‎Modifications:

Anbuser: /* Thermocycling Conditions */

2016-06-14T19:51:22Z

‎Thermocycling Conditions

Anbuser: /* Reaction Setup: */

2016-06-14T19:47:09Z

‎Reaction Setup:

Anbuser: /* Thermocycling Conditions */

2016-06-14T19:46:36Z

‎Thermocycling Conditions

Anbuser: Created page with " == Reaction Setup: == {| class="wikitable" !|'''STEP''' !|'''TEMPERATURE (°C)''' !|'''TIME''' |- |'''10 µM Forward Primer''' |2 µL |0.4 µM |- |'''10 µM Reverse Primer'..."

2016-06-14T19:45:37Z

Created page with " == Reaction Setup: == {| class="wikitable" !|'''STEP''' !|'''TEMPERATURE (°C)''' !|'''TIME''' |- |'''10 µM Forward Primer''' |2 µL |0.4 µM |- |'''10 µM Reverse Primer'..."

New page

== Reaction Setup: ==

{| class="wikitable"
!|'''STEP'''
!|'''TEMPERATURE (°C)'''
!|'''TIME'''
|-
|'''10 µM Forward Primer'''
|2 µL
|0.4 µM
|-
|'''10 µM Reverse Primer'''
|2 µL
|0.4 µM
|-
|'''Template DNA'''
|Y (1ng)
|1 ng
|-
|'''LongAmp Taq 2X Master Mix'''
|25 µL
|1X
|-
|'''Nuclease-free Water'''
|21- Y
|
|}

'''Final Volume = 50µL'''

Notes:

• LongAmp Taq 2X Master Mix can be used to amplify templates of up to 30kb long.

• LongAmp Taq 2X Master Mix can be stored at -20°C, however, it is only stable for 15 freeze-thaw cycles. For more frequent use, NEB recommends storing it at 4°C.

• Remember to first dilute primers in nuclease-free water if starting with a higher concentration

• Do not exceed 50 µL final volume

== Thermocycling Conditions==

{| class="wikitable"
!|'''REAGENT'''
!|'''VOLUME'''
!|'''FINAL CONC.'''
|-
|'''Initial Denaturation'''
|94
|1 min
|-
|'''30 Cycles''' ''denature''
''anneal''

''extend''
|94
45-60*

65
|20 sec
40 sec

50 sec per kb
|-
|'''Final Extension'''
|65
|10 min
|-
|'''Hold'''
|4-22
|
|}

← Older revision		Revision as of 20:09, 14 June 2016
Line 75:		Line 75:


−	To find annealing temperature, follow steps on the NEB Tm Calculator [http://tmcalculator.neb.com/#!/]	+	To find annealing temperature, follow steps on the NEB Tm Calculator, [http://tmcalculator.neb.com/#!/] which should yield a temperature 5°C below the Tm of the lower primer
−

	== Modifications ==		== Modifications ==

← Older revision		Revision as of 19:53, 14 June 2016
Line 78:		Line 78:


−	== Modifications: ==	+	== Modifications ==

	• If template is GC rich, a longer initial denaturation of 2-4 minute is recommended		• If template is GC rich, a longer initial denaturation of 2-4 minute is recommended

← Older revision		Revision as of 19:53, 14 June 2016
Line 1:		Line 1:

−	== Reaction Setup: ==	+	== Reaction Setup ==

	{\| class="wikitable"		{\| class="wikitable"

← Older revision		Revision as of 19:52, 14 June 2016
Line 110:		Line 110:
	\|		\|
	\|}		\|}
		+
		+	For more information, visit the NEB LongAmp Protocol [https://www.neb.com/protocols/2012/09/05/protocol-for-longamp-taq-2x-master-mix-m0287]

← Older revision		Revision as of 19:53, 14 June 2016
Line 31:		Line 31:

	Notes:		Notes:
		+
	• LongAmp Taq 2X Master Mix can be used to amplify templates of up to 30kb long.		• LongAmp Taq 2X Master Mix can be used to amplify templates of up to 30kb long.

@@ Line 27: / Line 27: @@
+|
 |}
 '''Final Volume = 50µL'''
 Notes:
 •	LongAmp Taq 2X Master Mix can be used to amplify templates of up to 30kb long.

@@ Line 59: / Line 59: @@
 ''extend''
 |94
--60*
+-60
@@ Line 65: / Line 65: @@
 sec
 sec per kb
 |-

@@ Line 3: / Line 3: @@
 {| class="wikitable"
-!|'''STEP'''
+!|'''REAGENT'''
-!|'''TEMPERATURE (°C)'''
+!|'''VOLUME (µL)'''
-!|'''TIME'''
+!|'''FINAL CONC.'''
 |-
 |'''10 µM Forward Primer'''