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        Gene Knockout with Conventional Mutagens

        互联网

        1315

         

        Gene Knockout with Conventional Mutagens

        Leon Avery

        ENG (enriched nematode growth medium): 
        1.  in a 6 L flask add:
         3800 ml DH2O
         20 g. bactopeptone
         4 g yeast extract
         12 g. NaCl
         4 ml 5mg/ml cholesterol in EtOH
        stir with a large stir bar until dissolved.
        
        2. Add 120 g. agar and stir well.
        
        3. Cover w/ Al foil, autoclave liq. cycle 30'
           (turn on the 70C water bath before the agar is autoclaved)
        
        4. Equilibrate temp. in water bath.
        
        5. Just before pouring the plates add (sterile solutions) with
           stirring:
         4 ml 1M CaCl2
         4 ml 1M MgSO4
         100 ml 1M KHPO4 (pH6)
                4 ml 200 mg/ml streptomycin
         1 ml 40 mg/ml nystatin in DMF (add dropwise)
        
        AENG (agarose ENG):
        Same recipe as ENG, except replace 3% agar with 2% agarose, and you
        probably won't want to make so much.
        
        Making a grid:
        
        1. Inoculate one or more 10 cm ENG plates with 10 N2 L4
           hermaphrodites.  Four or five days later, when there are many
           gravid adults, prepare eggs by the alkaline hypochlorite method.
           Leave the eggs in M9 after the final wash.
        
        2. Put the tube of eggs in M9 on a rocker (or something else that will
           aerate them) overnight.
        
        3. Count the healthy L1s in a 10 ul aliquot of the suspension.  (I use
           a 10 ul microcap to spread them on an unseeded 6 cm plate.)  Based
           on the count, put 5000 healthy L1s on each of 10 10 cm ENG plates.
           These will be your P0s.
        
        4. When the P0s have reached L4, harvest them in 3 ml M9.  (You'll
           probably have to spin them down and transfer them to new M9 to get
           the volume that low.)
        
        5. Dissolve 20 ul EMS in 1 ml M9.  Add this solution to your P0s.
        
        6. Aerate for 4 hours.
        
        7. Spin down, transfer to 4 ml fresh M9.
        
        8. Put 5000 P0s on each of 10 10 cm ENG plates.  Allow them to become
           gravid adults with lots of eggs.  (I do this at 15C, since the L4s
           will become gravid adults inconveniently soon at 20C.)
        
        9. Harvest all the plates and prepare eggs by alkaline
           hypochlorite.  Aerate overnight.
        
        10. Count the healthy F1 L1s in a 10 ul aliquot.  There will be a lot
           of sick or dead ones (their parents were mutagenized) -- don't
           count these.
           Determining numbers of F1s
        
        
        11. Put 1250 F1 L1s on each of 110 6 cm AENG plates.
           F1s per plate
        
        
        12. After 3 days at 20C most of the F1s should be gravid adults.
           Count the gravid adults on one or two of your plates.  There should
           be about 1000 (less than 1250, because many of the F1s didn't grow
           up or were sterile).
           F1s per plate
        
        
        13. After another 2 days (5 days total) the plates have starved, and
           almost all the worms on them are L1s.  Harvest 96 of the plates
           with 5 ml M9 each.  Discard any extra plates -- they're just in
           case some of your plates got moldy, or otherwise didn't work.
           Harvesting the plates
        
        
        14. Count a 10 ul aliquot from a few of the tubes.  You should have
           recovered between 100,000 and 200,000 F2 L1s from each plate.
           F2s per plate
        
        
        15. Let the tubes sit on your bench until the worms have settled to
           the bottom.  with a Pasteur pipet attached to vacuum, suck off
           liquid so that you have about 1.5 ml left in each tube.  (This is
           the step I worry about -- I'm afraid if the worms sit at the bottom
           of the tube too long they may go anaerobic and die.  I do plates in
           batches, so that they don't have to sit too long, and mix the tubes
           from the earlier batches occasionally while working on the later
           ones.)
        
        16. For this step, I use sterile 1.2 ml tubes in an 8x12
           microtiter-spaced rack.  You want three racks of 96, two for worms
           to be recovered later, and one for DNA preps.  Put 0.5 ml of 2x
           freezing solution in each of the tubes of the two worm racks.
           Then, for the first of your 96 tubes, put 0.5 ml into the A1
           position of each of the three racks.  For the second, put 0.5 ml
           into the A2 position, etc.
        
        17. Put the two worm racks into a styrofoam box in a -80C freezer and
           leave overnight.  You can freeze the worms for the DNA preps now,
           or go ahead and do the DNA preps immediately.
        
        18. Let the worms settle to the bottoms of the tubes, and suck off all
           the liquid you can.
           DNA preps
        
        
        19. Add 250 ul fresh lysis solution (200 mM NaCl, 100 mM Tris-HCl pH
           8.5, 50 mM EDTA, 0.5% SDS, 100 ug/ml proteinase K) to each tube.
        
        20. 50C, 1h.  (This is shorter and cooler than the typical incubations
           used for DNA preps.  It works fine, and long incubations at high T
           are mutagenic.)
        
        21. Prepare DNA from each tube by phenol:sevag extraction, sevag
           extraction, and EtOH precipitation.  Redissolve in 150 ul TER (10
           mM Tris-HCl pH 8, 1 mM EDTA, 1 ug/ml RNAase A.  Store at -80C.
        
        22. Use 2 ul of 1:5 diluted DNA in each detection reaction.
        
        Mutation detection protocols:
        
        I have used three different protocols for detecting mutations.  The
        first is for detecting point mutations in a restriction site.  I won't
        describe this, since I don't currently think it's the best way to
        knock out a gene.  The second method is that commonly used for
        detecting deletion of a Tc1 by relying on the much greater efficiency
        of standard PCR in amplifying shorter fragments.  I call this method
        "primer approximation", since it relies on the deletion approximating
        (bringing close together) the primers.  The third method asks for the
        deletion of a cluster of restriction enzyme sites.  It has the
        theoretical advantage that it may detect small deletions that don't
        much change the efficiency of amplification.  Whether this is a
        practical advantage is not clear.
        
        I do not use nested primers.  I think this reduces the false positive
        rate, but it does mean the primers have to be pretty good, since you
        need to be able to amplify from as little as 10 molecules to a good
        strong band.  Therefore, before screening with any primer pair, I
        first run a series of reactions inoculated with 0, 10, 1000, and
        100,000 wild-type genomes, and run under long PCR conditions
        (basically the conditions for site deletion below).  I make 25-mer
        primers with 40-60% GC, using the Whitehead Institute primer program
        to help find them and avoid primer-dimers, etc.  Most of the pairs
        work.
        
        Mutation detection by primer approximation:
        
        Solution A:
            0.4 ul 5 U/ul Taq polymerase
            5   ul 10x PCR buffer (100 mM Tris pH 8.3, 500 mM KCl, 20 mM MgCl2)
            0.5 ul 10 mg/ml acetylated BSA (NEB)
            1   ul 10 uM left primer
            1   ul 10 uM right primer
            1   ul 10 mM dNTPs (10 mM each of dATP, dCTP, dGTP, dTTP)
           39.1 ul water
           -------
           48   ul
        
        Put 48 ul A in each of 96 thin-wall 200 ul tubes.  Add 2 ul 1:5
        diluted DNA to each tube.  Run PCR program (MJR PTC-200):
        
            Control method: CALCULATED
            1: 92 degrees forever, beep.
            2: 92 degrees, 55 sec.
            3: 92 degrees, 5 sec.
            4: 65 degrees, 30 sec (adjust for the Tm of your primers).
            5: 68 degrees, 1 min.
            6: go to step 3 for 39 more cycles.
            7: 68 degrees, 4 min.
            8: 4 degrees forever, beep.
        
        Start the PCR machine and wait for the block to heat up.  Now put the
        tubes (kept on ice) in the block.  The solution in thin-wall tubes
        will heat up very fast when placed in a preheated block: this is
        almost as good as a hot start.  Push the proceed button on the PCR
        machine once all the tubes are loaded.
        
        Add 10 ul 6X gel loading buffer and run 12 ul on 1% gel.
        
        
        Mutation detection by site deletion:
        
        In this example, the enzyme BstBI is used.  You need to use a
        thermophilic enzyme, since cooling the reaction down to 37C for the
        second digestion has disastrous effects on PCR.  You also want an
        enzyme that will work pretty well in PCR buffer, and that is pretty
        cheap.  There are a whole series of enzymes from Bacillus
        stereothermophilus that meet these criteria.
        
        Solution A:
            0.25 ul 20 U/ul BstBI, NEB
            1 ul 10x NEBuffer 4
            0.1 ul 10 mg/ml acetylated BSA, NEB
            6.65 ul water
            ------
            8.0 ul
        
        Solution B:
            0.01 ul 5 U/ul Pwo polymerase
            0.09 ul 5 U/ul Taq polymerase
            3.5 ul 10x PCR-NEB4 (75 mM tris pH 8.6, 500 mM KCl)
            0.35 ul 10 mg/ml AcBSA
            1 ul 10 uM primer 1
            1 ul 10 uM primer 2
            1 ul 10 mM dNTPs
            28.05 ul water
            --------
            35.0 ul
        
        Solution C:
            0.3 ul 5 U/ul Taq polymerase
            0.5 ul 20 U/ul BstBI
            0.5 ul 10x PCR-NEB4
            0.05 ul 10 mg/ml AcBSA
            3.65 ul water
            -------
            5.0 ul
        
        LAMD45 PCR program (CALC mode, MJR PTC-200):
            1 92C forever, beep
            2 92C, 1 min
            3 65C, 30 sec (annealing temp)
            4 68C, 5 min
            5 65C forever, beep (T in steps 5 & 6 is digestion T for enzyme)
            6 65C, 30 min
            7 92C, 5 sec
            8 65C, 30 sec
            9 68C, 2 min
            10 go to step 7 for 8 more cycles
            11 92C, 5 sec
            12 65C, 30 sec (annealing temp)
            13 68C, 2 min + 10 sec/cycle
            14 go to step 11 for 34 more cycles
            15 72C, 10 min
            16 4C forever, beep
        
        1.  Mix 2 ul DNA solution (intended to be about 100,000 genomes) with
            8 ul A.  Incubate 65C, 10 min.
        
        2.  Add 35 ul B to each tube.  Keep everything on ice while you do
            this.
        
        3.  Start the PCR machine.  When it reaches 92C (step 1), transfer the
            tubes from the ice to the block.  Once all the tubes are in the
            block, tell the machine to proceed.
        
        4.  At step 5, the machine will halt again at 65C.  Leaving the tubes
            in the block, add 5 ul C to each.  When finished, tell the machine
            to proceed.
        
        6.  Run on 1% agarose gel.
        

         

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