Cloning

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{{back to protocols}}
{{back to protocols}}
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=General Procedure=
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==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.  
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=Materials=
+
==Materials==
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==Equipment==
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===Equipment===
*gas burner
*gas burner
*rotating plate
*rotating plate
*aluminium rack for PCR tubes
*aluminium rack for PCR tubes
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== Media and plates ==
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=== Media and plates ===
1.2- Reagents
1.2- Reagents
*[[LB]] (Luria-Bertami) agar plates
*[[LB]] (Luria-Bertami) agar plates
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**DW
**DW
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=Procedure=
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==Procedure==
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==Enriching the insert==
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===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]]
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==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]].
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==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]].
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===Reagents===
+
====Reagents====
*[[T4 DNA ligase]]
*[[T4 DNA ligase]]
*10x T4 DNA Ligase Buffer
*10x T4 DNA Ligase Buffer
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*Purified, linearized insert (likely in H2O or EB)
*Purified, linearized insert (likely in H2O or EB)
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===Method===
+
====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
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#Store at -20°C.
#Store at -20°C.
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==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]].
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===Reagents===
+
====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]]
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*ß-mercaptoethanol (ß-MAE)
*ß-mercaptoethanol (ß-MAE)
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===Method===
+
====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]]
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=Critical steps=
+
==Critical steps==
-
=Troubleshooting=
+
==Troubleshooting==
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=Notes=
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==Notes==
-
=See Also=
+
==See Also==
===Cloning Software===
===Cloning Software===
{{#dpl:category=cloning|category=software}}
{{#dpl:category=cloning|category=software}}

Revision as of 21:01, 12 January 2012

back to protocols

Contents

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

1.2- Reagents

  • LB (Luria-Bertami) agar plates
  • IPTG stock solution (100 nM)
  • SOC medium
  • DYT medium
  • 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

  1. Mix reagents for a 10 μL reaction.
  2. Let reaction sit at Room Temperature for 30 minutes OR overnight at 16°C
  3. Denature ligase at 65°C for 10 minutes.
  4. Store at -20°C.

Transformation

Transformation is the process of introducing foreign DNA (e.g plasmids, BAC) into a bacterium. See Bacterial transformation.

Reagents

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

Protocols

General

Lab-specific protocols


Discussion

You can discuss this protocol.

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