para II.EE del nivel Secundaria Ayacucho 9
5,578Total General
2.6 ADQUISICIÓN DE ACTIVOS NO FINANCIEROS 0.00 0.00 0.00 0.00 0.00
2. Centrifuge 1.5 ml of the overnight culture in a 2 ml eppendorf tube for 2 min 3. Wash the cells once with demineralized water
4. Resuspend the pellet in 0.5 ml solution A + lysozyme (5 mg/ml) 5. Incubate 10 min at 55 °C
6. Add 20 µl proteinase K (20 mg/ml), mix by inversion
7. Add 25 µl 10% SDS, mix thoroughly by inversion so that lysis can occur 8. Incubate at 60°C, at least for 1 hr (solution should become clear) 9. Add 250 µl demineralized water
10. Add 250 µl phenol, mix by inversion 11. Add 250 µl chloroform, mix by inversion 12. Centrifuge 2 min in eppendorf centrifuge 13. Transfer the water phase to a clean tube 14. Repeat the steps 10 to 13
15. Add 500 µl chloroform, mix by inversion 16. Centrifuge 1 min
17. Transfer 500 µl of the water phase to a clean tube
18. Add 50 µl 3 M NaAc ph 5.2 and 500 µl 2-propanol, mix by inversion until precipitated- DNA is visible
19. Centrifuge 5 min
20. Discard the supernatant and wash the DNA pellet with 70% ethanol (remove ethanol thoroughly, but do not dry the pellet)
21. Dissolve the DNA pellet in 200 µl TE + 5µl RNase (10 mg/ml)
1.3 Chromosomal DNA (for enzyme reactions)
This method is suitable for recombinant-DNA technique, for example the isolation of chromosomal DNA for the construction of gene banks (shotgun cloning).
Procedure 1. Grow an overnight culture in 100 ml TY medium (III.B.6); 37°C; aerate, or shake at 200 RPM, in an 1 l Erlenmeyer, or flask.
2. Collect the cells by centrifugation (6000 RPM, 10 min)
3. Resuspend the pellet in 2 ml solution A (Birnboim; VI.3.b) + lysozyme (2 mg/ml) 4. Incubate 30 min at 37°C
5. Add sarkosyl to a final concentration of 1% (Sarkosyl = Oramix; stock 30%) and add 10 µl RNase of a 10 mg/ml stock solution.
7. Add SDS to a final concentration of 0.5% and add 10 µl proteinase K of a 20 mg/ml solution (make a fresh solution).
8. Incubate 30 min at 37°C
9. Add 0.2 ml 60% PAS , mix (PAS: para-aminosalicytate) 10. Incubate 5 min, RT
11. Add 1 volume phenol/chloroform (1 : 1 mixture), mix well
Remark: If the phenol is defrozen the upper phase is the water phase. Take care to pipette the lower phenol phase (use a micro pipette or a pipetting balloon).
12. Centrifugate, 5 min, 10.000 RPM (separation of the water and phenol phase) 13. Collect the upper water phase containing the DNA with a 1 ml micropipette
14. Repeat step 11-13 twice (or more) until there is no more protein (white inter phase) present.
15. Add 1 volume chloroform and centrifugate 2 min. at 10.000 RPM. 16. Collect the upper water phase.
17. Dialyse 24 hour (two refreshments of the wash buffer) each time against 0.5 litre T10E1 (IV.10)
18. Determine the concentration of the DNA (VI.6 Dische reaction).
IV.2 Bacteriophage DNA
2.1. The preparation of a sterile bacteriphage stock
The purity of the starting-point material (phage and host) is very important for the preparation of good lysates that can be used for the isolation of DNA. Before making a big lysate you should have a sterile stock of the phage. This stock is prepared in the following way:
a. Plate lysate
1. Make an entplate with separate plaques on an suitable indicator.
2. Isolate with an sterile pasteur pipette 1 plaque and resuspend this plaque in 1ml diluted salts (SZ; III.B.3).
3. Filter with a membrane filter pore width 0,45 µm.
4. Plate out 0.1ml of the undiluted filtrate on ± 6 plates TY (+10 mM MgCl2). 5. Incubate over night at 37°C, which causes confluent lysis.
6. Per plate add 1.5 ml TY-bouillon (+ 10-2 M MgCl
2) and resuspend the top layers in
this TY with a bacterium spreader.
7. Collect the suspensions in transformation flask and remove the cells and cellular debris through centrifugation for 10 min 10000g.
8. Collect the supernatants and filter them with a membrane filter pore width 0,45 µm. 9. Determine the phage titer (IV.8.d)
10. Store the lysate at 4°C.
b.. Liquid lysate
1. Make an overnight culture of the indicator in TY medium +10 mM MgCl2. Dilute the
overnight culture 100 x in fresh medium and incubate 1 hour at 37°C (moderate aeration).
2. To 6 ml culture add 1 ml of the filtered plaque solution (see step 1-3 of method 1: plate lysate)
3. Incubate at 37°C with good aeration or shaking until lysis occurs (2-4 hour). 4. Remove the cells and cellular debris through centrifugation for 10 min 10000g. 5. Collect the supernatants and filter them with a membrane filter pore width 0,45 µm. 6. Determine the phage titre (IV.8.d)
7. Store the lysate at 4°C.
2.2 Large lysates (used for DNA isolations)
The procedures for the making of large lysates (1-2 litre) are different for the most phages. Use in each case a sterile stock of the phage (VI.2.a). After lysis all further steps in the isolation procedures are done in the same way. See 2c, 2d and 2e.
a. SPPI
In this case a special overnight culture is needed that can be made in the following way: 1. Take 50 ml TY-bouillon (+ 4x10-2 M MgCl
2) in a 100 ml bottle; and inoculate with a
suitable host (MCB) and incubate in an shaking water bath over night at 100 RPM (37°C).
2. A thin overnight culture is obtained the next morning. Take 2 litre TY-bouillon ( + 4x10-2 M MgCl
2) and dilute the overnight culture 1:50; aerate strongly and grow for 2
hours at37°C.
3. Infect with 5x105 pfu/ml (from a sterile stock; see a) and incubate at 37°C until
complete lysis occurs(2 à 4 hours).
4. If the lysis is not complete the culture can be put for several hours or overnight at 37°C, without aeration.
b. H1
1. Dissolve an overnight culture 1:50 in 1 or 2 litre TY medium, supplemented with MgCl2 (5x10-3 M)
2. Incubate at 37°C with strong aeration to A450 = ± 1,0
3. Infect with ± 108 pfu/ml, from a sterile stock (per litre you need 1011 pfu's. Keep this in
mind when you prepare the stock (see a)).
4. Incubate again at 37°C with strong aeration. If there is too much foam add some drops sterile anti-foam.
5. Lysis occurs after 2-4 hour.
2.3 Continued purification of lysate’s
Lysates of all B.subtilis phages can be purified in the following way:
1. Determine the titre before you continue with the next steps (titer determination: IV.8.d.1).
2. Add DNAase to 10 µg/ml, MgCl2 to 10-2 M), and RNase to 10 µg/ml and incubate
during 30 min at 37°C (mix well).
Remark: this step is only necessary if you need very pure DNA. Omit this step if you need the DNA for transfections.
3. Remove the cell debris and cells by centrifugation for 10 min at 6000 RPM in a high- speed centrifuge.
4. Add NaCl to 0,5 M and PEG (polyethyleenglycol 6000) to 10%. After the PEG and NaCl have dissolved, place the solution minimal 3 hours at 0°C, or during the night in the cold room. Proteins including the phages will precipitate.
5. Collect the precipitate by centrifugation 10 min at 9000 RPM (high-speed centrifuge); Resuspend the pellets in a as small as possible volume TBT buffer (III.A.26)
(maximal 8 ml if the initial volume is 1 or 2 litre lysate).
Remark: In some cases (e.g. the isolation of transfecting DNA) no further purification is necessary and you can continue with step e (isolation of bacteriophage DNA). If further purification is needed ( e.g. enzyme reactions, separation of DNA strains en physical-chemical characterisation of DNA), continue the procedure with step d.
2.4 Purification and concentration of bacteriophage using CsCl density gradient
centrifugation.
Bacteriophage purified with the protocol 2c is not pure; It contains proteins and a lot of cellular material. Further purification is often required for example in a discontinue gradient of CsCl.
a. Procedure
1. Make the following solutions (TBT: III.A.26):
56,24% (w/w) CsCl, high quality Lab Reagent, in TBT (ρ=1,700) 45,41% (w/w) CsCl, high quality Lab Reagent, in TBT (ρ=1,500) 31,24% (w/w) CsCl, high quality Lab Reagent, in TBT (ρ=1,300)
Watch it!: w/w = weight percent; so 56,24% means 5,624 g CsCl and 4,376 g (= ml) TBT (and not fill up to10 ml)
2. Take the appropriate centrifuge tubes for the SW41Ti rotor (Beckman) 3. Fill up with the use of a pipette, an discontinue gradient:
Bottom layer ± 4,0 ml CsCl, ρ=1,700
Middle layer ± 4,0 ml CsCl, ρ=1,500 Top layer ± 4,0 ml CsCl, ρ=1,300
4. Carefully layer 4 à 5 ml phage suspension on the gradient (with a pipette) 5. Equilibrate the tubes (maximal difference is 0.05 g)
6. Centrifugate minimal 3 hour at 24.000 RPM (or over night) in the SW41Ti rotor
7. After centrifugation, remove the blue-grey tyndallising phage band with a pasteur-pipette as concentrated as possible.
8. Titre the phage (usually1012 - 1013 pfu/ml) (IV.8.d)
9. You can store the phage in the concentrated CsCl solution at 4°C.