(→BP reactions) |
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== BP Reactions == | == BP Reactions == | ||
| − | • Donor Vectors | + | ''• Donor Vectors'' |
| + | |||
| + | ''• PCR Amplification of DNA'' | ||
| − | + | Primers for PCR are designed as described in the Invitrogen Multi-Site Gateway Manual. This results in primers that are quite long (regularly >50 bases), but we have not had difficulty performing PCR with these primers. | |
| + | We have used two different polymerases for PCR: Tth (GeneAmp XL PCR Kit; Applied Biosystems) and Phusion (NEB). Both are proofreading polymerases that can amplify long pieces of DNA, although for particularly difficult and/or long promoters, Phusion has worked better. For each, PCR was performed in a 50 ul reaction. | ||
| − | |||
| + | ''• Purification of PCR products'' | ||
| − | + | The entire PCR reaction (50 ul) is loaded onto an agarose gel. The appropriate band is excised and DNA purification performed using the Qiagen Qiaquick Gel Extraction Kit (for DNA fragments <10 kb; for larger fragments, use the QIAEX Gel Extraction Kit). Elute the DNA in 30 ul (the smallest recommended volume). The concentration of DNA is calculated using a spectrophotometer; the DNA will be quite dilute and not terribly clean (usually between 10-80 ng/ul, and OD 260/280 ~1.4-1.6). | |
| + | Do not let the gel-purified DNA sit in the freezer for too long before using in the recombination reaction. In practice, we go straight into the recombination reaction; a better stopping point is to freeze either the entire PCR reaction or the gel slice before purification. We have found that storing the DNA in the freezer for even a couple of days decreases the efficiency of recombination. | ||
| − | • Transformation, Plasmid Prep, and Diagnostic Digests | + | |
| + | ''• BP Recombination Reactions'' | ||
| + | |||
| + | The recombination reaction is performed as described in the Invitrogen Multi-Site Gateway Manual. An equimolar amount of the appropriate donor vector and purified PCR product (commonly 50-100 femtomoles) are combined with TE and BP clonase enzyme mix in a final volume of 10 ul. This reaction is allowed to incubate overnight at room temperation, however, we have found (as the manual also suggests) that as little as 2 hours can be enough. This reaction is typically very robust. | ||
| + | |||
| + | |||
| + | ''• Transformation, Plasmid Prep, and Diagnostic Digests'' | ||
| + | |||
| + | The BP reaction is treated with Proteinase K and transformed. Typically, 2 ul of the 10 ul reaction is sufficient. The Invitrogen Manual recommends OneShot TOP10 cells, but cells of this high competence are not necessary. Subcloning efficiency cells and also homemade competent cells have worked well. A note: the clear/opaque difference in colonies applies only to transformants from the LR reaction, not this (the BP) reaction. | ||
== LR reactions == | == LR reactions == | ||
Revision as of 03:41, 8 February 2007
Note: The Invitrogen Multi-Site Gateway Manual contains all of the basic information necessary to understand and perform Gateway recombination reactions. The protocols here reflect the methods that the Chien Lab has adopted after much trial and error.
BP Reactions
• Donor Vectors
• PCR Amplification of DNA
Primers for PCR are designed as described in the Invitrogen Multi-Site Gateway Manual. This results in primers that are quite long (regularly >50 bases), but we have not had difficulty performing PCR with these primers.
We have used two different polymerases for PCR: Tth (GeneAmp XL PCR Kit; Applied Biosystems) and Phusion (NEB). Both are proofreading polymerases that can amplify long pieces of DNA, although for particularly difficult and/or long promoters, Phusion has worked better. For each, PCR was performed in a 50 ul reaction.
• Purification of PCR products
The entire PCR reaction (50 ul) is loaded onto an agarose gel. The appropriate band is excised and DNA purification performed using the Qiagen Qiaquick Gel Extraction Kit (for DNA fragments <10 kb; for larger fragments, use the QIAEX Gel Extraction Kit). Elute the DNA in 30 ul (the smallest recommended volume). The concentration of DNA is calculated using a spectrophotometer; the DNA will be quite dilute and not terribly clean (usually between 10-80 ng/ul, and OD 260/280 ~1.4-1.6).
Do not let the gel-purified DNA sit in the freezer for too long before using in the recombination reaction. In practice, we go straight into the recombination reaction; a better stopping point is to freeze either the entire PCR reaction or the gel slice before purification. We have found that storing the DNA in the freezer for even a couple of days decreases the efficiency of recombination.
• BP Recombination Reactions
The recombination reaction is performed as described in the Invitrogen Multi-Site Gateway Manual. An equimolar amount of the appropriate donor vector and purified PCR product (commonly 50-100 femtomoles) are combined with TE and BP clonase enzyme mix in a final volume of 10 ul. This reaction is allowed to incubate overnight at room temperation, however, we have found (as the manual also suggests) that as little as 2 hours can be enough. This reaction is typically very robust.
• Transformation, Plasmid Prep, and Diagnostic Digests
The BP reaction is treated with Proteinase K and transformed. Typically, 2 ul of the 10 ul reaction is sufficient. The Invitrogen Manual recommends OneShot TOP10 cells, but cells of this high competence are not necessary. Subcloning efficiency cells and also homemade competent cells have worked well. A note: the clear/opaque difference in colonies applies only to transformants from the LR reaction, not this (the BP) reaction.
LR reactions
need to explain image:
