Cloning: Difference between revisions
From OpenWetWare
Jump to navigationJump to search
mNo edit summary |
m (→Transformation) |
||
| (3 intermediate revisions by the same user not shown) | |||
| Line 1: | Line 1: | ||
{{back to protocols}} | {{back to protocols}} | ||
=General Procedure= | ==General Procedure== | ||
This should be a [[Help:Consensus protocol|consensus protocol]]. See the bottom of this article for specific protocols. | This should be a [[Help:Consensus protocol|consensus protocol]]. See the bottom of this article for specific protocols. | ||
=Materials= | ==Materials== | ||
==Equipment== | ===Equipment=== | ||
*gas burner | *gas burner | ||
*rotating plate | *rotating plate | ||
*aluminium rack for PCR tubes | *aluminium rack for PCR tubes | ||
== Media and plates == | === Media and plates === | ||
'''Reagents''' | |||
*[[LB]] (Luria-Bertami) agar plates | *[[LB]] (Luria-Bertami) agar plates | ||
*[[IPTG]] stock solution (100 nM) | *[[IPTG]] stock solution (100 nM) | ||
| Line 22: | Line 22: | ||
**DW | **DW | ||
=Procedure= | ==Procedure== | ||
==Enriching the insert== | ===Enriching the insert=== | ||
The insert can come from another vector or loaded from a PCR product. PCR product should be clean by various [[Purification of DNA|purification methods]] and there exists several protocols for [[PCR]] | The insert can come from another vector or loaded from a PCR product. PCR product should be clean by various [[Purification of DNA|purification methods]] and there exists several protocols for [[PCR]] | ||
==Restriction Digest== | ===Restriction Digest=== | ||
Depending on the ends required for ligation, the ends of the insert and the vector may need to be prepared. For cloning methods like TA cloning, the Taq polymerase will add on the appropriate sticky ends. For other methods such as blunt-end ligation and sticky-end ligation, you will need digested vector and insert. See [[Restriction digest]]. | Depending on the ends required for ligation, the ends of the insert and the vector may need to be prepared. For cloning methods like TA cloning, the Taq polymerase will add on the appropriate sticky ends. For other methods such as blunt-end ligation and sticky-end ligation, you will need digested vector and insert. See [[Restriction digest]]. | ||
==Ligation== | ===Ligation=== | ||
The process of ligating or joining ends of DNA together. This will take linear strand(s) of DNA and circularize the product to result in the desired plasmid. For more details see [[DNA ligation]]. | The process of ligating or joining ends of DNA together. This will take linear strand(s) of DNA and circularize the product to result in the desired plasmid. For more details see [[DNA ligation]]. | ||
'''Reagents''' | |||
*[[T4 DNA ligase]] | *[[T4 DNA ligase]] | ||
*10x T4 DNA Ligase Buffer | *10x T4 DNA Ligase Buffer | ||
| Line 38: | Line 38: | ||
*Purified, linearized insert (likely in H2O or EB) | *Purified, linearized insert (likely in H2O or EB) | ||
'''Method''' | |||
#Mix reagents for a 10 μL reaction. | #Mix reagents for a 10 μL reaction. | ||
#Let reaction sit at Room Temperature for 30 minutes OR overnight at 16°C | #Let reaction sit at Room Temperature for 30 minutes OR overnight at 16°C | ||
| Line 44: | Line 44: | ||
#Store at -20°C. | #Store at -20°C. | ||
==Transformation== | ===Transformation=== | ||
Transformation is the process of introducing foreign DNA (e.g plasmids, BAC) into a bacterium. See [[Bacterial transformation]]. | Transformation is the process of introducing foreign DNA (e.g plasmids, BAC) into a bacterium. See [[Bacterial transformation]]. | ||
'''Reagents''' | |||
*[[Competent cells]]: XL2-Blue (Stratagene 200150). | *[[Competent cells]]: XL2-Blue (Stratagene 200150). | ||
**Learn how to make your own by [[Preparing_chemically_competent_cells]] | **Learn how to make your own by [[Preparing_chemically_competent_cells]] | ||
| Line 53: | Line 53: | ||
*ß-mercaptoethanol (ß-MAE) | *ß-mercaptoethanol (ß-MAE) | ||
'''Method''' | |||
==Screening== | ===Screening=== | ||
Either a [[Restriction_digest| check digest]] or [[Colony PCR]] will be a good way of screening your colonies. If the plasmid is of the correct size, submit for [[DNA sequencing]] | Either a [[Restriction_digest| check digest]] or [[Colony PCR]] will be a good way of screening your colonies. If the plasmid is of the correct size, submit for [[DNA sequencing]] | ||
=Critical steps= | ==Critical steps== | ||
=Troubleshooting= | ==Troubleshooting== | ||
=Notes= | ==Notes== | ||
=See Also= | ==See Also== | ||
===Cloning Software=== | ===Cloning Software=== | ||
{{#dpl:category=cloning|category=software}} | {{#dpl:category=cloning|category=software}} | ||
Revision as of 18:03, 12 January 2012
| back to protocols | ||
General Procedure
This should be a consensus protocol. See the bottom of this article for specific protocols.
Materials
Equipment
- gas burner
- rotating plate
- aluminium rack for PCR tubes
Media and plates
Reagents
- Other consumables
- gloves
- sterile (???) plates
- pH paper
- pasteur pipettes
- DW
Procedure
Enriching the insert
The insert can come from another vector or loaded from a PCR product. PCR product should be clean by various purification methods and there exists several protocols for PCR
Restriction Digest
Depending on the ends required for ligation, the ends of the insert and the vector may need to be prepared. For cloning methods like TA cloning, the Taq polymerase will add on the appropriate sticky ends. For other methods such as blunt-end ligation and sticky-end ligation, you will need digested vector and insert. See Restriction digest.
Ligation
The process of ligating or joining ends of DNA together. This will take linear strand(s) of DNA and circularize the product to result in the desired plasmid. For more details see DNA ligation.
Reagents
- T4 DNA ligase
- 10x T4 DNA Ligase Buffer
- Purified, linearized vector (likely in H2O or EB)
- Purified, linearized insert (likely in H2O or EB)
Method
- Mix reagents for a 10 μL reaction.
- Let reaction sit at Room Temperature for 30 minutes OR overnight at 16°C
- Denature ligase at 65°C for 10 minutes.
- Store at -20°C.
Transformation
Transformation is the process of introducing foreign DNA (e.g plasmids, BAC) into a bacterium. See Bacterial transformation.
Reagents
- Competent cells: XL2-Blue (Stratagene 200150).
- Learn how to make your own by Preparing_chemically_competent_cells
- SOC medium
- ß-mercaptoethanol (ß-MAE)
Method
Screening
Either a check digest or Colony PCR will be a good way of screening your colonies. If the plasmid is of the correct size, submit for DNA sequencing
Critical steps
Troubleshooting
Notes
See Also
Cloning Software
- Digital Tools & Resources
- ApE - A Plasmid Editor (software review)
- Gene Construction Kit software review
- MCDS
Protocols
General
- Digital Tools & Resources
- Cloning and sequencing
- Cloning Checklist
- General Cloning Protocol
- Cloning Protocol
Lab-specific protocols
- Knight:TOPO TA cloning
- Wikiomics:Cloning in silico
- Wikiomics:Site Directed Mutagenesis
- Duffy:LIC Cloning
- Rutgers:DNA Ligation
- Rutgers:Transformation
- Milo:No background cloning protocol
Discussion
You can discuss this protocol.
