By D. Marks

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3. It is important to add the PEG 3350 first to protect the yeast cells from the high concentration of LiOAc. 4. At this point the cells are fragile and need to be suspended gently with a pipette; do not vortex. 5. This ensures that the cells are in the logarithmic growth phase during induction. 6. An alternative method for separating cell lysates from glass beads is to collect them into 15 mL Falcon tubes. To do that, cut a round hole in the cap of a Falcon tube, pierce the bottom of the breaking tube with a needle, and insert it into the cut cap.

Com/resources/bioinformaticstools 6. Serial-Cloner : http://serialbasics. html 7. edu/jorgensen/wayned/ape/ Molecular and structural biology websites 8. edu/mpstruc/ 9. com/nmr/MPNMR. html Academic expression plasmid resources 10. Protein Science Initiative. 1 Designing Constructs for Expression 1. Before starting molecular cloning experiments, check if your target MP is already available in an expression vector (see Protein Science Initiative Web site) and search the literature to see if your MP target or related proteins have been produced in recombinant systems (see Note 1).

If possible engineer dua-ribosome-binding site (RBS) expression vectors like pET-Duet (Novagen) so that you can clone a fluorescent protein (FP) gene downstream of your target MP gene. 1). FP fusion with your target MP is also an option developed successfully by several laboratories [12]. 2 Selecting the Optimal Expression Vector/ Bacterial Host Selecting the right combination of vector/bacterial host is an essential step to achieve the optimal production of your MP (see Note 2). The following rules apply to the T7 RNAP-based expression system: 1.

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