Richard Lab:Amplified insert assembly: Difference between revisions
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==Procedure== | ==Procedure== | ||
Your two parts will be thusly labeled "insert" and "vector" | Your two parts will be thusly labeled "insert" and "vector" and will be initially contained on separate plasmids. The basic steps are as follows: | ||
#Miniprep both "insert" and "vector" from their respective cultures (30 mins). | #Miniprep both "insert" and "vector" from their respective cultures (30 mins). | ||
#PCR the insert plasmid (2 hrs) | #PCR the "insert" plasmid (This will take about 2 hrs, but start the vector digest right away). | ||
##Use a high-fidelity polymerase (eg. pfu Turbo or Deep Vent). | ##Use a high-fidelity polymerase (eg. pfu Turbo or Deep Vent). | ||
##Use the same primers you use for colony PCR (Annealing Temp of 55°C). | ##Use the same primers you use for colony PCR (Annealing Temp of 55°C). | ||
| Line 26: | Line 26: | ||
#Digest insert for 1 hour (adding DpnI along with the other restriction endonucleases). | #Digest insert for 1 hour (adding DpnI along with the other restriction endonucleases). | ||
#Add Antarctic Phosphatase to the "vector" digest and incubate until Insert Digest is done. | #Add Antarctic Phosphatase to the "vector" digest and incubate until Insert Digest is done. | ||
#Ligate with 3μL of vector and 3μL of insert in a 20ml Ligation. | #Kill all reactions by incubating for 20 mins at °C. | ||
#Ligate with 3μL of vector and 3μL of insert in a 20ml Ligation (Don't forget to kill the ligation). | |||
#Transform. | #Transform. | ||
#Plate. | |||
#Celebrate. | #Celebrate. | ||
This entire process should take around 6 hours as many steps overlap. | |||
==Notes== | ==Notes== | ||
Revision as of 16:11, 11 January 2011
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Overview
This is what Mike calls "Standard Assembly 2.0" and is a method of "bio-bricking" two biological parts (i.e. pieces of DNA together. For more information on bio-bricking see this link. This method combines the ease and speed of triple antibiotic assembly with the fidelity of standard assembly. Major benefits of this assembly method over other assembly methods include:
- no gel electrophoresis or extractions
- The ability to insert small (i.e. invisible on a gel) parts.
- No need to fool with multiple antibiotic resistances.
- No having to make construction vectors.
This protocol is typically used to do bio-brick assembly with restriction sites consisting of the following configuration:
Ocassionally other enzymes (e.g. BamHI or HindIII) are used to make protein fusions.
Procedure
Your two parts will be thusly labeled "insert" and "vector" and will be initially contained on separate plasmids. The basic steps are as follows:
- Miniprep both "insert" and "vector" from their respective cultures (30 mins).
- PCR the "insert" plasmid (This will take about 2 hrs, but start the vector digest right away).
- Use a high-fidelity polymerase (eg. pfu Turbo or Deep Vent).
- Use the same primers you use for colony PCR (Annealing Temp of 55°C).
- Digest vector for 2 hours.
- Purify the PCR product.
- Digest insert for 1 hour (adding DpnI along with the other restriction endonucleases).
- Add Antarctic Phosphatase to the "vector" digest and incubate until Insert Digest is done.
- Kill all reactions by incubating for 20 mins at °C.
- Ligate with 3μL of vector and 3μL of insert in a 20ml Ligation (Don't forget to kill the ligation).
- Transform.
- Plate.
- Celebrate.
This entire process should take around 6 hours as many steps overlap.
Notes
- The DpnI eliminates any background from the insert PCR.
- The phosphatase eliminates any background vector.
References
Contact
or instead, discuss this protocol.
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