Mutagenesis - Making Defective-Mu Pools in Salmonella
Jeff Lawrence (X25)
The hopping is achieved by providing Mu transposase in cis on the transducing DNA. The adjacent Mud-1 has a ts repressor, so it must be grown at 30° at all times.
1. Streak out donor strain on LB-Kan plates at 30°:
TT10288 has MudJ for transcriptional fusions
TT10381 has MudK for translational fusions
2. Pick five colonies and grow overnight cultures at 30°. Make a transducing lysate on each.
3. On the same overnight cultures, transduce each to prototrophy with an LT2 lysate on E-Glucose. Print transduction plate to LB-kan.
4. Abandon lysates on strains with significant KanR prototrophic transductants; these cultures have had the Mu element hop in the donor lysate.
5. Transduce the recipient strain to KanR with one of these lysates. If the recipient has an intact his operon region, 50% of the transductants will be His- transductants (searches for auxotrophs are done on E-Glu-Kn-his plates). Transduce the recipient cell with a dilution series of the phage lysate to find conditions yielding 1000-3000 drug-resistant colonies per plate.
6. Scale the proper transduction up to generate a pool of 50,000 - 100,000 independent hops. This is 20 to 100 plates. Don't let the colonies get too big; pin-head size is good.
7. Add 2 mL of 1xE Salts to each plate and use a spreader to suspend all of the colonies.
8. Pool the cells as desired into 50 mL centrifuge tubes using Pasteur pipettes.
9. Centrifuge cells and resuspend in 1xE Salts + 10 mM EGTA. Repeat centrifugation.
10. Resuspend cells in LB + 10 mM EGTA. Freeze most cells.
11. Add 0.5 mL cells to 10 mL LB with appropriate antibiotic. Grow overnight.
12. Prepare 50 mL of P22 lysate on the 10 mL of overnight culture in separate tubes.
13. Spin down cell debris and pool lysate over chloroform.
Last Update: Thursday, 19-Jun-2014 11:54:48 PDT
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