NanoBio: Site-Directed Mutagenesis: Difference between revisions
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(New page: == Site-Directed Mutagenesis == * Use QuikChange's Mult-Site Directed Mutagenesis Kit to do site-directed mutagenesis, even if only one site is to be mutated. * Design and order PAGE-purif...) |
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== Site-Directed Mutagenesis == | == Site-Directed Mutagenesis == | ||
This protocol describes how make deletions, mutations, and additions to an existing plasmid. Site-directed mutagenesis is frequently used to remove unwanted BioBrick or BioFusion restriction sites from a part's sequence. I recommend using QuikChange's MultiSite Directed Mutagenesis Kit to do site-directed mutagenesis, even if only one site is to be mutated. | |||
* Design and order PAGE-purified primer(s). [http://www.stratagene.com/ | |||
===Materials=== | |||
* QuikChange Multi Site Directed Mutagenesis Kit (Stratagene, catalog # 200515) | |||
* template DNA | |||
* mutagenesis primers (1 per region to be changed) | |||
===Procedure=== | |||
* Design and order PAGE-purified primer(s). [http://www.stratagene.com/tradeshows/feature.aspx?fpId=118 Stratagene's website] has a nice program to help you design primers. Briefly, your primers must: | |||
** be between 25-45 bp long | ** be between 25-45 bp long | ||
** have the site to be mutated approximately in the middle of the primer, with 10-15 bp flanking this site | ** have the site to be mutated approximately in the middle of the primer, with 10-15 bp flanking this site | ||
** have a melting temperature greater or equal to 75C | ** have a melting temperature greater or equal to 75C | ||
***Note: Use [http://www.stratagene.com/QPCR/tmCalc.aspx Stratagene's Tm calculator] to determine the T<sub>m</sub> of your primers; it is different from Vector NTI's calculated T<sub>m</sub>. | |||
** have a GC content of at least 40% | ** have a GC content of at least 40% | ||
** (only if using multiple primers): all bind to the same template strand | ** (only if using multiple primers): all bind to the same template strand | ||
* See the [http://www.stratagene.com/ | |||
* See the [http://www.stratagene.com/tradeshows/feature.aspx?fpId=146 instruction manual] for additional details. | |||
* If this is the first time you're doing this procedure, do the positive control included in the kit. | |||
* When doing the site-directed mutagenesis, follow the instructions in the manual, with these changes: | * When doing the site-directed mutagenesis, follow the instructions in the manual, with these changes: | ||
**Do two pcr reactions: one with 1.0 uL of template DNA, and a second with 0.5 uL template DNA. | **Do two pcr reactions: one with 1.0 uL of template DNA, and a second with 0.5 uL template DNA. | ||
| Line 15: | Line 25: | ||
**From 500 uL of SOC + transformants, plate 50uL. Centrifuge the remaining cells and media. Leaving the pelleted cells intact, remove all but ~150 uL of media. Re-suspend the cells, then spread them on a second plate. Essentially, you are plating all the cells from remaining 450 uL transformation mixture, but just decreasing the volume that you are spreading on the plate to ~150 uL. | **From 500 uL of SOC + transformants, plate 50uL. Centrifuge the remaining cells and media. Leaving the pelleted cells intact, remove all but ~150 uL of media. Re-suspend the cells, then spread them on a second plate. Essentially, you are plating all the cells from remaining 450 uL transformation mixture, but just decreasing the volume that you are spreading on the plate to ~150 uL. | ||
* In our experience, this procedure works well. There are [http://openwetware.org/wiki/Site-directed_mutagenesis other protocols] around if you run into problems. | * In our experience, this procedure works well. There are [http://openwetware.org/wiki/Site-directed_mutagenesis other protocols] around if you run into problems. | ||
*'''[[User:CarolineAjo-Franklin|CAjoF]] 17:59, 31 March 2008 (EDT)''':. | |||
Latest revision as of 15:01, 31 March 2008
Site-Directed Mutagenesis
This protocol describes how make deletions, mutations, and additions to an existing plasmid. Site-directed mutagenesis is frequently used to remove unwanted BioBrick or BioFusion restriction sites from a part's sequence. I recommend using QuikChange's MultiSite Directed Mutagenesis Kit to do site-directed mutagenesis, even if only one site is to be mutated.
Materials
- QuikChange Multi Site Directed Mutagenesis Kit (Stratagene, catalog # 200515)
- template DNA
- mutagenesis primers (1 per region to be changed)
Procedure
- Design and order PAGE-purified primer(s). Stratagene's website has a nice program to help you design primers. Briefly, your primers must:
- be between 25-45 bp long
- have the site to be mutated approximately in the middle of the primer, with 10-15 bp flanking this site
- have a melting temperature greater or equal to 75C
- Note: Use Stratagene's Tm calculator to determine the Tm of your primers; it is different from Vector NTI's calculated Tm.
- have a GC content of at least 40%
- (only if using multiple primers): all bind to the same template strand
- See the instruction manual for additional details.
- If this is the first time you're doing this procedure, do the positive control included in the kit.
- When doing the site-directed mutagenesis, follow the instructions in the manual, with these changes:
- Do two pcr reactions: one with 1.0 uL of template DNA, and a second with 0.5 uL template DNA.
- Digest with DpnI for at least 5 hrs., or preferably overnight, rather than for 1 hour.
- XL-10 Gold Cells can be transformed in eppendorf tubes using SOC media, rather than in Falcon tubes with NZY+ media.
- After adding the DpnI-digested pcr reaction to the XL-10 Gold cells, incubate on ice for 45 min - 1hr, instead of 30 min.
- From 500 uL of SOC + transformants, plate 50uL. Centrifuge the remaining cells and media. Leaving the pelleted cells intact, remove all but ~150 uL of media. Re-suspend the cells, then spread them on a second plate. Essentially, you are plating all the cells from remaining 450 uL transformation mixture, but just decreasing the volume that you are spreading on the plate to ~150 uL.
- In our experience, this procedure works well. There are other protocols around if you run into problems.
- CAjoF 17:59, 31 March 2008 (EDT):.
