McClean:Oligonucleotide phosphorylation, Annealing and Ligation

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Contents

Overview

This is a protocol for oligo phosphorylation, annealing for cloning.

Materials

  • Forward oligo
  • Reverse oligo
  • 1X TE buffer
  • 10X Annealing Buffer
  • 10X T4 DNA Ligase Buffer
  • T4 Polynucleotide Kinase

Stock Solutions

1X TE buffer

  • This is a very simple solution, so we only need a one line description of how to make it.

10X Annealing Buffer

  • Recipe for 4ml, add
                     400ul 1M Tris pH 8
                     80ul 0.5M EDTA pH8
                     800ul 2.5M NaCl
                     2720ul Water


Protocol

  1. Resuspend oligos to a stock concentration of 100uM in 1X TE buffer. Store oligo stocks at -20oC when not using.
  2. To a PCR tube, add
                    2 ul of the proper Top or Bottom strand oligo 
                    2 ul of 10X T4 DNA Ligase Buffer 
                    1 ul of T4 Polynucleotide Kinase
                    15 ul of water
  Mix well and spin down. Oligo final concertration is 10 uM.
                    
  1. Incubate the PCR tube in the thermocycler with the program at 37 degree for 60 minutes, then at 65 degree for 20 minutes then end.
  2. Annealing the phosphorylated FW and RV Oligos:
                                                FW oligo 5ul
                                                RV oligo 5ul
                                    10X Annealing Buffer 5ul
                                                   Water 35ul
   Mix well and spin down. The final oligo concertration is 1uM.
  1. Incubate the PCR tube in the thermocycler with the program at 95 degree for 3 minutes, then -1 degree every cycle for 60 cycles, at 25 degree for 5 minutes then end.
  2. Calculate the molarity of vector and annealed oligos to be used for the ligation.
For Vector: (__ ng vector)/(670 ng/nmol-base pair x __ base pairs x 10 exp-6 L) = ___ nM (nmol/L)
For Oligo: 1uM = 1000nM 
  1. Use proper amount of vector and oligo to ligate and transform to DH5 alpha 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.

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References

http://gcat.davidson.edu/mediawiki-1.15.0/index.php/Annealing_Oligos_for_Cloning

Contact

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