Endy:Site-directed Mutagenesis
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| + | ''Back to [[Site-directed mutagenesis]]'' | ||
==Primer Design== | ==Primer Design== | ||
| - | *See [[Designing | + | *See [[Designing primers | this page]] for general advice and software suggestions for primer design. |
*Primers should contain the mutations to be introduced but should anneal to the sequence to be mutated. You will need a forward and reverse primer. | *Primers should contain the mutations to be introduced but should anneal to the sequence to be mutated. You will need a forward and reverse primer. | ||
*Primers should be between 25 and 45 base-pairs in length. | *Primers should be between 25 and 45 base-pairs in length. | ||
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==Experimental Protocol== | ==Experimental Protocol== | ||
| - | *See the [http://www.stratagene.com/manuals/200518.pdf Stratagene manual] for the suggested protocols. | + | *See the [http://www.stratagene.com/manuals/200518.pdf Stratagene manual] for the suggested protocols. The notes below are suggested modifications to this protocol that may be useful depending on what you are trying to do. |
| - | + | ||
*It makes sense to dilute the mutagenic primers to the same concentration as the control primers used in the [http://www.stratagene.com Stratagene] protocol, i.e. 100ng/μl. | *It makes sense to dilute the mutagenic primers to the same concentration as the control primers used in the [http://www.stratagene.com Stratagene] protocol, i.e. 100ng/μl. | ||
*Leon suggested that attempting to modify three consecutive bases is difficult. He suggested using 4-5 more cycles than recomended by [http://www.stratagene.com Stratagene]. | *Leon suggested that attempting to modify three consecutive bases is difficult. He suggested using 4-5 more cycles than recomended by [http://www.stratagene.com Stratagene]. | ||
*Leon also recommended letting the DpnI digestion run for 2-3 hours. | *Leon also recommended letting the DpnI digestion run for 2-3 hours. | ||
| - | *Doing a [[PNK Treatment of DNA Ends|PNK step]] on the primers should boost | + | *Doing a [[PNK Treatment of DNA Ends|PNK step]] on the primers should boost efficiency. |
| + | *It is not necessary to use XL1-Blue cells. Any highly competent cells should be ok. | ||
Current revision
Back to Site-directed mutagenesis
Primer Design
- See this page for general advice and software suggestions for primer design.
- Primers should contain the mutations to be introduced but should anneal to the sequence to be mutated. You will need a forward and reverse primer.
- Primers should be between 25 and 45 base-pairs in length.
- The mutation(s) should be in the middle of the primers.
- Mutation efficiency is greatly influenced by the purity of the primers. Its worth getting purified primers. No need for 5' phosphorylation.
- See the Stratagene manual for more detailed information.
Experimental Protocol
- See the Stratagene manual for the suggested protocols. The notes below are suggested modifications to this protocol that may be useful depending on what you are trying to do.
- It makes sense to dilute the mutagenic primers to the same concentration as the control primers used in the Stratagene protocol, i.e. 100ng/μl.
- Leon suggested that attempting to modify three consecutive bases is difficult. He suggested using 4-5 more cycles than recomended by Stratagene.
- Leon also recommended letting the DpnI digestion run for 2-3 hours.
- Doing a PNK step on the primers should boost efficiency.
- It is not necessary to use XL1-Blue cells. Any highly competent cells should be ok.
