Making MudJ, K, F and Mud-Cam Pools 10/5/91
The general idea is that a tranducing lysate is grown on a strain with the mini Mud in hisD and full Mud-1 (hisA9941::Mud-1) right next door. This lysate is used to transduce some recipient to Kan resistance. The P22 can't package more than the mini Mud and the near end of the Mud-1, which contains the MuA and B genes ( the transposase genes). The recipient of the cross can become Kan resistant by the legitimate recombination of the mini-Mud into his material (if this homology is provided in the recipient) or by a transposition of the mini Mud off of the transducing partical to the chromosome, the Mu transposase being provided in cis on the same fragment. Really the only tricky part is assuring oneself that the donor is correct; since the Mud-1 is essentially a wild-type Mu with a ts repressor (MuC) there are sometimes extra mini-Muds in the background. If the mutagenizing lysate is grown on such a strain many of the resulting KanR colonies will have this Mud at the same spot by recombination and the level of actual mutagenesis, unique independent insertions, will be much lower than you thought.
1) Streak the donor strain from the frozen onto NB Kan plates and grow at 30oC for single colonies.
MudJ Transciptional fusions TT10288
MudF Entier lac operon This doesn't exist as a his
construct?(Use TT8350 and
TT12116, like a Tn10 hop).
MudK Translational fusions TT10381
Mud-Cam No lac homology TT16528
2) Pick five to ten colonies and grow an overnight of each in NB at 30oC.
3) Make a transdusing lysate on each o/n as usual, but at 30oC!
4) Also with these overnights, check for second Muds in the background. Do this by using LT2 lysate to tranduce each o/n culture to prototrophy on E glu plates, at 30oC. Now print to E glu Kan plates. All the prototrophs should be Kan sensitive. In practice a few percent will always be KanR; if more are, that lysate should be discarded.
Note that this would't identify a second Mud that caused auxotrophy.
5) Use these lysates to mutagenize your chosen strain. These transductants can be screened directly or can be pooled. The rest of this protocol is specific to making a pool. If the recipient strain has an intact his region 50% of the KanR colonies well be recombinants at his, the rest will be hops. This will have to be remembered in all subsequent screens of the pool. (Searches for auxotrophs would be done by printing NB Kan and E glu his Kan). Or use as a recipient a deletion that removes his (TT5998 his-3050del). Only the region between the mini-Mud and the Mud-1 need be deleted.
6) Use a dilution series of the mutagenizing lysate to determine the correct concentration of phage. 1000-3000 KanR colonies per plate is good. Remember the problems of scaling up. Remember that NB is not all that rich, you may want to supplement it through out the following procedure.
7) Scale up. A good pool has between 50,000 and 100,000 independent hops in it. This is 50 to 100 plates.
8) Don't let the colonies get too big, pin head size is good.
9) Add 2ml of 1xE salts 10mM ETGA to each plate and use a spreader to suspend all the colonies.
10) 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.
11) Spin down cells.
12) Resuspend in a equal volume of 1xE salts 10mM EGTA and spin down again.
13) Resuspend in an equal volume of NB 10mM EGTA. Most of these cells can be frozen away in DMSO vials as is.
14) Dilute above cells 1:20 into NB 10mM ETGA and grow over-night. 20 to 50ml of final lysate is a good goal, that requires 4 to 10 ml of o/n.
15) Use thses cells to make a tranducing lysate: Spin over-night cells out of NB ETGA and resuspend in an equal volume 1xE salts.
16) Wash once in an equal volume 1xE salts, and resuspend in an equal volume of 1xE salts.
17) 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.
18) Spin down cells and debris and put phage pool over CHCl3.
Last Update: Thursday, 19-Jun-2014 12:32:26 PDT
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