Golden Gate Assembly Protocol for BsmB1 - Revision history

Kyroland at 12:38, 14 June 2018

2018-06-14T12:38:29Z

Macampbell at 16:43, 8 June 2016

2016-06-08T16:43:24Z

Macampbell at 16:39, 8 June 2016

2016-06-08T16:39:43Z

Eckdahl at 19:26, 1 June 2015

2015-06-01T19:26:11Z

Eckdahl at 19:25, 1 June 2015

2015-06-01T19:25:31Z

Macampbell at 14:42, 13 October 2014

2014-10-13T14:42:53Z

Macampbell at 14:41, 13 October 2014

2014-10-13T14:41:43Z

Macampbell at 14:39, 13 October 2014

2014-10-13T14:39:44Z

Eckdahl at 19:48, 20 May 2013

2013-05-20T19:48:26Z

ToEckdahl: Golden Gate Assembly Protocol for BsmB1

2013-03-06T21:24:21Z

Golden Gate Assembly Protocol for BsmB1

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Golden Gate Assembly Protocol for BsmB1
GGA mixture contains:
1 µL (50 ng) Plasmid
1 µL Promoter
1 µL 10X Promega Ligase Buffer
6 µL dH2O 0.5 µL BsmB1 high fidelity (HF) restriction enzyme
9.5 µL volume
Place tubes into 55°C water bath for 15 minutes. Add 0.5 µL T4 DNA Ligase to the mixture.
Turn on the PCR machine. Put tube into machine. Program it for the following cycles:
• 20 cycles
• 55°C for 10 minutes
• 37°C for 1 minute
• 16°C for 1 minute
• 22°C holding temperature
This DNA ligation is ready for transformation. You can increase the number of cycles to 30 if you want to increase yield. However, we have gotten good success with as few as 5 cycles.
Transformation of GGA BsmB1
For each reaction done you will need an agar plate and 50µL of competent cells mixed with competent cell buffer. Mix the 50µL of cell mixture with the GGA product. Place the entire content of the tube on the plate, spread and incubate.

@@ Line 8: / Line 8: @@
 :1 µL 10X Promega Ligase Buffer
-:6 µL dH<sub>2</sub>O 0.5 µL BsmB1 high fidelity (HF) restriction enzyme
+:6 µL dH<sub>2</sub>O
 :9.5 µL volume

@@ Line 1: / Line 1: @@
-Golden Gate Assembly Protocol
+Golden Gate Assembly Protocol for BsmB1
 GGA mixture contains:
-:1 µL (50 ng) receiving plasmid
+:1 µL (50 ng) Plasmid
-:1 µL promoter or other insert at X ng/µL (to determine X ng of insert = (bp insert) (50 ng linearized plasmid-) ÷ (size of entire plasmid in bp)
+:1 µL promoter or other insert
-:1 µL 10X NEB Ligase Buffer
+:1 µL 10X Promega Ligase Buffer
-:6 µL dH<sub>2</sub>O
+:6 µL dH<sub>2</sub>O 0.5 µL BsmB1 high fidelity (HF) restriction enzyme
-:0.5 µL Restriction enzyme (BsmBI-HF, EcoRI-HF, PstI-HF, BsaI-HF, or BbsI)
+:9.5 µL volume
-: 0.5 µL T4 DNA Ligase from NEB to the mixture
+Place tubes into 55°C water bath for 15 minutes. Add 0.5 µL T4 DNA Ligase to the mixture.
-:10 µL total volume
+Turn on the PCR machine. Put tube into machine. Program it for the following cycles:
-Turn on the thermocycler machine. Put tube into machine. Program it for the following cycles:
+:• 55°C for 10 minutes
 :• 37°C for 1 minute
 :• 16°C for 1 minute
 :• 22°C holding temperature
@@ Line 32: / Line 28: @@
 This DNA ligation is ready for transformation. You can increase the number of cycles to 30 if you want to increase yield. However, we have gotten good success with as few as 5 cycles.
-Transformation of GGA
+Transformation of GGA BsmB1
 For each reaction done you will need an agar plate and 50µL of competent cells mixed with competent cell buffer. Mix the 50µL of cell mixture with the GGA product. Place the entire content of the tube on the plate, spread and incubate.

@@ Line 2: / Line 2: @@
 GGA mixture contains:
-:1 µL (50 ng) Plasmid
+:1 µL (50 ng) receiving plasmid
-:1 µL promoter or other insert
+:1 µL promoter or other insert at X ng/µL (to determine X ng of insert = (bp insert) (50 ng linearized plasmid-) ÷ (size of entire plasmid in bp)
-:1 µL 10X Promega Ligase Buffer
+:1 µL 10X NEB Ligase Buffer
 :6 µL dH<sub>2</sub>O
@@ Line 12: / Line 12: @@
 :0.5 µL Restriction enzyme (BsmBI-HF, EcoRI-HF, PstI-HF, BsaI-HF, or BbsI)
-: 0.5 µL T4 DNA Ligase to the mixture
+: 0.5 µL T4 DNA Ligase from NEB to the mixture
 :10 µL total volume
-Turn on the PCR machine. Put tube into machine. Program it for the following cycles:
+Turn on the thermocycler machine. Put tube into machine. Program it for the following cycles:
-:• 20 cycles
+:• 20 cycles of
-:• 55°C for 10 minutes
+:• 37°C for 1 minute
-:• 37°C for 1 minute
+:• 16°C for 1 minute
-:• 16°C for 1 minute
+:• 37°C for 15 minutes
 :• 22°C holding temperature

← Older revision		Revision as of 19:26, 1 June 2015
Line 14:		Line 14:
	: 0.5 µL T4 DNA Ligase to the mixture		: 0.5 µL T4 DNA Ligase to the mixture

−	:10 µL volume	+	:10 µL total volume

@@ Line 2: / Line 2: @@
 GGA mixture contains:
-µL (50 ng) Plasmid
+:1 µL (50 ng) Plasmid
-µL Promoter
+:1 µL promoter or other insert
-µL 10X Promega Ligase Buffer
+:1 µL 10X Promega Ligase Buffer
-µL dH<sub>2</sub>O 0.5 µL BsmB1 high fidelity (HF) restriction enzyme
+:6 µL dH<sub>2</sub>O 0.5 µL BsmB1 high fidelity (HF) restriction enzyme
-.5 µL volume
+:9.5 µL volume
 Place tubes into 55°C water bath for 15 minutes. Add 0.5 µL T4 DNA Ligase to the mixture.