Difference between revisions of "Protocols"

Revision as of 12:39, 16 February 2007

Note: the Invitrogen multisite Gateway manual contains all of the basic information necessary to understand and perform Gateway recombination reactions. The protocols described here reflect the optimized methods that the Chien lab has adopted after much trial and error. See also the Gateway tips on the Lawson lab website. Please report problems or questions on the Tol2kit blog.

As a first test, we suggest that you grow up entry clones and perform a test LR reaction with pDestTol2pA2 or pDestTol2CG2 to make an expression clone such as bactin2:EGFP-pA.

Growing up clones

Entry clones are kanamycin-resistant and can be transformed and grown in any standard E. coli strain.

However, note that destination vectors and donor vectors have an ampicillin resistance gene in the backbone, and both a ccdB suicide gene and a chloramphenicol resistance gene in the "gate" (the ccdB provides negative selection during the BP or LR reaction). Therefore these clones must be grown in ampicillin/chloramphenicol, in ccdB-tolerant cells (available from Invitrogen). In addition, we have found that certain destination vectors and donor vectors are prone to recombination or mutation, so at a minimum you should test each DNA prep by careful restriction analysis.

What you will need

coming soon: list of reagents you will need

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

Recombination Reactions
The recombination reaction is performed with slight modifications to the protocol in the Invitrogen Multi-Site Gateway Manual. Equimolar amounts of entry vectors (5', middle, and 3') and destination vector are combined with LR clonase buffer and enzyme mix. We standardly set up reactions with 20 femtomoles. The manual contains a protocol for a 20 ul reaction; we halve everything and set up 10 ul reactions. This saves enzyme and therefore, money. Other labs have found that quarter reactions (5 ul) work as well.

We always allow this reaction to go overnight at room temperature. The reaction tends to be less efficient than the BP reaction, likely because of the number of components involved.

Transformation, Plasmid Prep, and Diagnostic Digests
As with the BP reaction, the LR reaction is treated with Proteinase K and then transformed. We typically transform 3 ul of the 10 ul reaction, using Invitrogen OneShot TOP10 cells. Because this reaction is less efficient than the BP reaction, cells of this high competence are necessary. One other note is that when using these cells, it is recommended that after heat shock, the cells be shaken at 37 degrees for 1 hour. We typically shake the transformation for 1.5 hours, just to give the cells more time to grow before selection. Particular LR recombination reactions can be less efficient than others (see below), and we believe that giving the culture one more doubling time will increase the chance that the correct clone can be isolated.

This reaction is plated onto ampicillin plates; carbenicillin works as well. We do not plate the reaction before 3 pm, as satellite colonies can be a significant problem, obscuring the results of the reaction. Plates are removed from the 37 degree incubator first thing the next morning; this provides the best chance to distinguish clear from opaque colonies. If it is difficult to tell clear from opaque, looking at the plate in front of a dark background (we use a black refrigerator) will help. The image below shows examples of clear and opaque colonies on the same plate.

Plates can be left at room temperature until clear colonies are picked in the afternoon. We have found that clear colonies contain the correct clone >99% of the time, while opaque colonies never contain the correct clone. A reaction that has worked well will have a clear to opaque colony ratio of at least 3:1. However, as long as clear colonies can be identified, the correct clone will be isolated.