Difference between revisions of "Golden Gate Assembly Protocol for BsmB1"

From GcatWiki
Jump to: navigation, search
Line 1: Line 1:
Golden Gate Assembly Protocol for BsmB1
+
Golden Gate Assembly Protocol  
 
GGA mixture contains:  
 
GGA mixture contains:  
  
Line 8: Line 8:
 
:1 µL 10X Promega Ligase Buffer  
 
: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  
+
:0.5 µL Restriction enzyme (BsmBI-HF, EcoRI-HF, PstI-HF, BsaI-HF, or BbsI)
 +
 
 +
: 0.5 µL T4 DNA Ligase to the mixture
 +
 
 +
:10 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:  
 
Turn on the PCR machine. Put tube into machine. Program it for the following cycles:  
Line 28: Line 31:
 
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.  
 
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
+
Transformation of GGA  
 
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.
 
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.

Revision as of 17:25, 1 June 2015

Golden Gate Assembly Protocol GGA mixture contains:

1 µL (50 ng) Plasmid
1 µL promoter or other insert
1 µL 10X Promega Ligase Buffer
6 µL dH2O
0.5 µL Restriction enzyme (BsmBI-HF, EcoRI-HF, PstI-HF, BsaI-HF, or BbsI)
0.5 µL T4 DNA Ligase to the mixture
10 µL volume


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 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.