Making Mini-Tn10 Pools (Tn10dTet, Tn10dCanm and Tn10dKam)
To make these pools a recipient is provided with a plasmid carrying the Tn10 transposase. The donor carries the Tn10 derived transposon on an E. coli F', so that there is no homology to the Salmonella chromosome which it can use to recombine onto the Salmonella chromosome. When the transduction is done the drug resistant colonies have had a transposition event to rescue the transposon off of the transducing fragment. Each colony is a indepedent hop.
1) Move pNK972 (TT10427) into the recipient strain by transduction, selecting Amp.
2) Grow a phage lystate on the donor.
Tn10d-Tet TT10423
Tn10d-Cam TT10605
Tn10d-Kam TT10426
3) Use a dilution series of the lysate to determine the correct concentration of phage. 1000-3000 DrugR colonies per plate is good. Remember the problems of scaling up. Remember that NB is not all that rich (cys, ade, aro, etc.), you may want to supplement it throughout the following procedure.
4) Scale up. A good pool had between 50,000 and 100,000 independent hops in it. This is 50 to 100 plates.
5) Don't let the colonies get too big, pin head size is good.
6) Add 2ml of 1xE salts 10mM EGTA to each plate and use a spreader to suspend all the colonies.
7) Tilt the petri dishes and transfer the cell suspension to green-top tubes, or 50ml centrifuge tubes, with a pasture pipette. All the cells can be pooled at this point, or sub-pools of 10,000 each can be maintained.
8) Spin down cells.
9) Resuspend in an equal volume o 1xE salts 10mM EGTA and spin down again.
10) Resuspend in an equal volume of NB 10mM EGTA. Most of these can be frozen away in DMSO vials as is.
11) Dilute above cells 1:20 into NB 10mM EGTA and grow over-night. 20 to 50ml of final lysate is a good goal, that requires 4 to 10ml of o/n.
12) Use these cells to make a transducing lysate: Spin over-night cells out of NB EGTA and resuspend in an equal volume 1xE salts.
13) Wash once in an equal volume 1xE salts, and resuspend in an equal volumes of 1xE salts.
14) Mix with P22 phage broth as usual. Don't scale up, it doesn't work very well, use multiple 5ml cultures in green top tubes.
15) Spin down cells and debris and put phage pool over CHCl3.
Last Update: Thursday June 19 2014
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Eric Kofoid
eckofoid at ucdavis.edu