McClean:Oligonucleotide phosphorylation, Annealing and Ligation: Difference between revisions
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==Overview== | ==Overview== | ||
This is a protocol for oligo phosphorylation, annealing for cloning. | This is a protocol for oligo phosphorylation, annealing for cloning. | ||
==Materials== | ==Materials== | ||
* | * Forward oligo | ||
* | * Reverse oligo | ||
* | * 1X TE buffer | ||
* | * 10X Annealing Buffer | ||
* 10X T4 DNA Ligase Buffer | |||
* T4 Polynucleotide Kinase | |||
==Stock Solutions== | ==Stock Solutions== | ||
''' | '''1X TE buffer''' | ||
* This is a very simple solution, so we only need a one line description of how to make it. | * 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== | ==Protocol== | ||
# | # 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 | |||
==Notes== | ==Notes== | ||
<!-- Please paste this section "as is" into your protocol, and add notes to it if you have some!--> | <!-- Please paste this section "as is" into your protocol, and add notes to it if you have some!--> | ||
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==References== | ==References== | ||
http://gcat.davidson.edu/mediawiki-1.15.0/index.php/Annealing_Oligos_for_Cloning | |||
==Contact== | ==Contact== | ||
<!--Change the information below to your info if you add a new protocol--> | <!--Change the information below to your info if you add a new protocol--> | ||
*'''[[User: | *'''[[User:Ping Xu|Ping Xu]] 14:01, 20 July 2011 (EDT)''' | ||
or instead, [[Talk:{{PAGENAME}}|discuss this protocol]]. | or instead, [[Talk:{{PAGENAME}}|discuss this protocol]]. |
Revision as of 08:48, 24 October 2013
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
- 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
Notes
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.
References
http://gcat.davidson.edu/mediawiki-1.15.0/index.php/Annealing_Oligos_for_Cloning
Contact
- Ping Xu 14:01, 20 July 2011 (EDT)
or instead, discuss this protocol.