Corum:T4 Ligation

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Revision as of 18:03, 30 August 2012 by Sean P Corum (Talk | contribs)
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  • SC 15:16, 19 June 2012 (EDT):

Contents

Overview

Standard DNA ligation using NEB T4 ligase to form a vector.

Materials

For a 10 μL ligation reaction:

  • 5 μL ~100nM linker dsDNA (either hybridization product or digested, purified PCR product)
  • 0.5 μL ~10nM backbone dsDNA (gel extracted digestion product from donor plasmid)
  • 2 μL sterile ddH2O
  • 1 μL 10X ligation buffer
  • 1 μL 10mg/ml BSA
  • 0.5 μL T4 ligase

(Note: standard ratio of backbone to linker given here is ~1:100. This may need to be adjusted for non-standard conditions.)

Procedure

  1. Add the reaction components together in order listed above. Mix gently and spin.
  2. Include -linker control reaction (sub sterile ddH2O for linker).
  3. Incubate 16 °C overnight (8 hr minimum).
  4. Transform 2.5 μL rea(or more, if necessary, to obtain an acceptable amount of colonies) into chemically competent cells.

Notes

Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input!

  1. List troubleshooting tips here.
  2. You can also link to FAQs/tips provided by other sources such as the manufacturer or other websites.
  3. Anecdotal observations that might be of use to others can also be posted here.

Please sign your name to your note by adding '''*~~~~''': to the beginning of your tip.


  1. *SC 18:03, 30 August 2012 (EDT): Removed 1mg/ml BSA component from protocol, per the fact that it is not recommended in the NEB protocol.

References

  1. New England Biolabs

Contact

or instead, discuss this protocol.

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