Difference between revisions of "Golden Gate Assembly Protocol for BsmB1"
(Golden Gate Assembly Protocol for BsmB1) |
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Golden Gate Assembly Protocol for BsmB1 | Golden Gate Assembly Protocol for BsmB1 | ||
GGA mixture contains: | GGA mixture contains: | ||
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1 µL (50 ng) Plasmid | 1 µL (50 ng) Plasmid | ||
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1 µL Promoter | 1 µL Promoter | ||
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1 µL 10X Promega Ligase Buffer | 1 µL 10X Promega Ligase Buffer | ||
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6 µL dH2O 0.5 µL BsmB1 high fidelity (HF) restriction enzyme | 6 µL dH2O 0.5 µL BsmB1 high fidelity (HF) restriction enzyme | ||
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9.5 µL volume | 9.5 µL volume | ||
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Place tubes into 55°C water bath for 15 minutes. Add 0.5 µL T4 DNA Ligase to the mixture. | Place tubes into 55°C water bath for 15 minutes. Add 0.5 µL T4 DNA Ligase to the mixture. | ||
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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: | ||
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• 20 cycles | • 20 cycles | ||
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• 55°C for 10 minutes | • 55°C for 10 minutes | ||
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• 37°C for 1 minute | • 37°C for 1 minute | ||
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• 16°C for 1 minute | • 16°C for 1 minute | ||
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• 22°C holding temperature | • 22°C holding temperature | ||
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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. | ||
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Transformation of GGA BsmB1 | 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. | 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 19:48, 20 May 2013
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.
