Cloning

From OpenWetWare

Revision as of 23:52, 29 December 2011 by Christopher C Vanlang (Talk | contribs)
Jump to: navigation, search
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

Ligation

Just 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.

Personal tools