Corum:T4 Ligation

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For a 10 μL ligation reaction:
For a 10 μL ligation reaction:
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* 7 μL linker dsDNA (either 100nM standard concentration linker hybridization product or standard digested and purified PCR product, which is usually ~100nM)
+
* 5 μL linker dsDNA (either 100nM standard concentration linker hybridization product or standard digested and purified PCR product, which is usually ~100nM)
-
* 0.5 μL backbone dsDNA (usually ~10 nM) (Note: ratio of backbone to linker should be 10-20:1. The specific amounts of linker and backbone dsDNAs may need to be adjusted to keep this ratio for non-standard concentrations)
+
* 0.5 μL backbone dsDNA (usually ~10 nM) (Note: ratio of backbone to linker should be 1:100. The specific amounts of linker and backbone dsDNAs may need to be adjusted to keep this ratio for non-standard concentrations)
 +
* 2 μL sterile ddH<sub>2</sub>O
* 1 μL 10X ligation buffer
* 1 μL 10X ligation buffer
* 1 μL 10mg/ml BSA
* 1 μL 10mg/ml BSA

Revision as of 11:35, 29 June 2012

  • 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 linker dsDNA (either 100nM standard concentration linker hybridization product or standard digested and purified PCR product, which is usually ~100nM)
  • 0.5 μL backbone dsDNA (usually ~10 nM) (Note: ratio of backbone to linker should be 1:100. The specific amounts of linker and backbone dsDNAs may need to be adjusted to keep this ratio for non-standard concentrations)
  • 2 μL sterile ddH2O
  • 1 μL 10X ligation buffer
  • 1 μL 10mg/ml BSA
  • 0.5 μL T4 ligase

Procedure

  1. Add the reaction components together in order listed above. Mix gently and spin.
  2. Incubate 16 °C overnight (8 hr minimum).

Notes

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  1. List troubleshooting tips here.
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References

Relevant papers, books, and websites

  1. New England Biolabs

Contact

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