Mutagenesis - Making Defective-Tn10 Pools in Salmonella
Jeff Lawrence (X20)
1. Prepare a recipient strain by transducing a plasmid bearing the Tn10 transposase into the desired background. Suitable plasmids include:
pNK972 in strain TT10427 bears the wild-type transposase
pNK2881 in strain TT17193 bears the ATS transposase
2. Prepare a P22 lysate on a donor strain. These strains bear defective Tn10 elements on E. coli F' plasmids and will not recombine into the S. typhimurium genome. Suitable strains include:
TT10423 bears Tn10dTc
TT10605 bears Tn10dCm
TT10426 bears Tn10dKn
TT18324 bears Tn10dGn
3. Transduce the recipient cell with a dilution series of the phage lysate to find conditions yielding 1000-3000 drug-resistant colonies per plate.
4. Scale the proper transduction up to generate a pool of 50,000 - 100,000 independent hops. This is 20 to 100 plates.
5. Don't let the colonies get too big; pin-head size is good.
6. Add 2 mL of 1xE Salts to each plate and use a spreader to suspend all of the colonies.
7. Pool the cells as desired into 50 mL centrifuge tubes using Pasteur pipettes.
8. Centrifuge cells and resuspend in 1xE Salts + 10 mM EGTA. Repeat centrifugation.
9. Resuspend cells in LB +10 mM EGTA. Freeze most cells.
10. Add 0.5 mL cells to 10 mL LB with appropriate antibiotic. Grow overnight.
11. Prepare 50 mL of P22 lysate on the 10 mL of overnight culture in separate tubes.
12. Spin down cell debris and pool lysate over chloroform.
Last Update: Thursday, 19-Jun-2014 11:54:48 PDT
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