pmkrein26-05-98

After identifying positive plaques on the initial screen, you must perform second and perhaps third screen.

 
N.B. Have NZY plates, SM medium, and top agar ready before starting.

1.) Identify positive plaques from initial screen. Choose plaques to propagate.
2.) Mark film with membrane/plate identification hole pokes.
3.) Align film to plate (hole pokes) on light box.
4.) Using blunt end of sterile glass pasture pipet poke through gel surrounding positive plaque.
5.) Place piece of gel containing phage into microfuge tube containing approx. 1ml SM media.
6.) Do the same for all positive plaques, placing into individual microfuge tube.
7.) Incubate gel piece (phage) in SM media 15-20 minutes at room temp with gentle agitaition (rocker).
8.) Prepare 1:10 dilutions of phage. Will plate 10-3, 10-4, 10-5 dilutions of phage. (10-3 should have far to many phage, while 10-5 should have very few plaques (=40). Thus 10-4 should be the ideal titer-- secondary and tertiary screens may have fewer phage, perhaps 10-1 best)
9.) Mix 100µl of Phage titer (each 10-3, 10-4, 10-5 dilutions) with 100µl P2392 cells grown in the presence of 10mM MgSO4. Incubate these with gentle agitation for 15-30minutes at 37 C. This allows the phage to bind, and inject their DNA into the P2392 E.coli.
10.) Mix with 4ml autoclaved Top agar (cooled to 45 -50 C), pour onto 100mm NZY plate. Tilt plate gently but quickly to coat plate surface. Top Agar will solidify within 5 seconds.
11.) Grow plates at 37 C for 8-10 hours. DO NOT OVERGROW. If can not monitor plates, grow for a few hours, place at 4 C and grow again following day. Ideally, want to grow until plaques are clearly seen-- thus suffictient phage particles to transfer and get a signal when screened, but are not so big that they are overrunning oneanother.
12.) After plaques are grown to suficient size, cool plates at 4 C for at least 1 hour.
13.) Cut nitrocellulose (nytran or hybond) filters to appropriate size, prepare enough to have duplicate filters for each plate.
14.) Select phage plate with best phage titer.
15.) Label each membrane with ball point pen, or lead pencil.
16.) carefully place dry membrane, labeled side UP on the plates. Be careful not to slide, or rotate membrane on plate. If there is some sliding, simply discard membrane and repeat.
17.) Using 18 guage needed (or pipet tip) poke holes through the membrane and agar, to identify orientation of phage lift.
18.) Incubate membranes on plate for 5-10 minutes.
19.) remove membrane, place phage side up (labeled side down) on bench paper. Allow membranes to completely dry (causes phage to BIND membrane). This should take approximately 10 minutes.
20.) Repeat lift with duplicate membranes, poke holes through membrane at same location as first membrane. This may be more difficult if holes in agar as small.
21.) After drying membranes completely, cut three strips of Whattman paper, sufficiently large to place membranes on.
22.) Soak the first strip with Denaturing solution. Soak with enough fluid to saturate the Whattman paper, but not so much that there are pools of solution on the surface of the paper.
23.) Place membranes PHAGE SIDE UP on the strip. Ensure that the denaturing solution is soaked into the membrane. Incubate this way for 2 minutes.
24.) Soak the second strip of Whattman paper in Neutralizing solution, as was done for denaturing solution. Transfer membranes to neutralizing solution as above. incubate for 2 mintues.
25.) Soak the final strip of Whattman in 2x SSC, transfer the filters as above.
26.) Take membranes on 2xSSC Whattman paper to Chris Brown's Lab and UV cross link to bind DNA to membranes. Alternately, membranes can be baked at 42 C overnight.
27.) Store membranes at 4 C in parafilmed petri dish.
28.) Screen at convineinece as was done in inital screen.

SOLUTIONS:

NZY Plates: (for propagation of bacteriophage)
21g NZY broth powder (Gilbco/BRL)
15g Bacto Agar.
Qs. to 1000ml.
Autoclave.
Plate as LB agar plates.
Top Agar:(for lambda phage)
10g Tryptone
2.5g NaCl
7g Bacto-Agar
Qs. to 1000ml, Autoclave.
Place in 50 C H2O bath until needed.
Suspension Medium (SM): (for phage resuspension; titer)
5.8g NaCl
2g MgSO4"7H2O
50ml 1M TRIS"Cl pH 7.5
0.01% gelatin (Difco)
Qs. to 1000ml. Heat to 50 C to suspend gelatin.
Denaturing Solution:
1.5M NaCl
0.5M NaOH
Neutralizing Solution:
1.5M NaCl
0.5M TRIS"Cl pH 8.0
20x SSC:
3M NaCl (175g/L)
0.3M Na3Citrate"2H2O (88g/L)
Adjust pH to 7.0 with 1M HCl
2x SSC:
Dilute 20x SSC 1:10 in ddH2O