Here are the specific reagents required for working with the Tol2Kit, including Invitrogen catalog numbers. We use the following antibiotic concentrations: ampicillin (100 ug/ml), kanamycin (50 ug/ml), and chloramphenicol (30 ug/ml), both for selection on plates as well as in liquid culture. We have also found that while propagating these vectors, it is crucial to pour plates of the desired antibiotic resistance it is not sufficient to pipet chloramphenicol solution onto a pre-poured ampicillin or kanamycin plate.įor donor vectors and destination vectors, which you will use repeatedly and which are tricky to grow up correctly, we recommend that you grow at least a midiprep or maxiprep scale. 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. Therefore these clones must be grown in ampicillin/chloramphenicol or kanamycin/chloramphenicol, in ccdB-tolerant cells (available from Invitrogen).

However, note that destination vectors have an ampicillin resistance gene in the backbone, donor vectors have a kanamycin resistance gene in the backbone, and both have a ccdB suicide gene and a chloramphenicol resistance gene in the 'gate' (the ccdB provides negative selection during the BP or LR reaction). If you run low, you can always just miniprep again. using Qiagen miniprep columns) are sufficient for the entry vectors you will use very little DNA for each LR reaction.

6.1 Preparation of capped transposase RNAĮntry clones are kanamycin-resistant and can be transformed and grown in any standard E.

5 Assembling sequences for expression clones.

4.3 Factors Affecting Reaction Efficiency.

4.2 Transformation, Plasmid Prep, and Diagnostic Digests.

3.5 Transformation, Plasmid Prep, and Diagnostic Digests.

Sequencher stored at room temperature