McClean:Oligonucleotide phosphorylation, Annealing and Ligation
This is a protocol for oligo phosphorylation, annealing for cloning.
- Forward oligo
- Reverse oligo
- 1X TE buffer
- 10X Annealing Buffer
- 10X T4 DNA Ligase Buffer
- T4 Polynucleotide Kinase
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
- Resuspend oligos to a stock concentration of 100uM in 1X TE buffer. Store oligo stocks at -20oC when not using.
- 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.
- 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.
- 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.
- 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.
- 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
Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input!
- List troubleshooting tips here.
- You can also link to FAQs/tips provided by other sources such as the manufacturer or other websites.
- 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.
- Ping Xu 14:01, 20 July 2011 (EDT)
or instead, discuss this protocol.