Protocol for DNA- DNA Hybridization

Protocol for DNA- DNA Hybridization:

1.     DNA fixing (for one Probe):

Ø  Add 60 µl 0.1 mg/ml all DNA Solution (Probe + Target) in 1.5 ml microcentrifuge tube.

Ø  Boil the tube for 5 min (90⁰C).

Ø  Immediately cool in Ice quickly.

Ø  Add 9.0 volume of PBSM buffer.

DNA Solution:

Ø  Dispense 0.1 ml DNA solution each in 96 well-microplate wells (96 wells).

Ø  Each well contains 1.0 µg of DNA.

Ø  Dispense the negative control in 96 well-microplate wells. (E.coli or Salmon Sperm DNA)

Ø  Cover the plates with aluminum foil.

Ø  Incubate at 37⁰C for 2 hrs. or 28⁰C for 3 hrs.

Ø  Discard the DNA Solution.

Ø  Wash with 0.2 ml 1X PBS (Remove unbound DNA).

Ø  Discard the PBS solution.

Ø  Dry at 60⁰C for 2 hrs. Or 45⁰C for overnight.

2.     Hybridization and Labelling of Probe:

Ø  Prepare 10 µl of Probe DNA (1 mg/ml= 1 µg/ µl).

Ø  Make or take prehybridization solution and hybridization solution from -20⁰C to the hybrid oven (37⁰).

Ø  Dispense 0.2 ml of prehybridization solution in microplate wells.

Ø  Incubate the wells at 37⁰C for 1 hr.

Photobiotin Labelling to the Probe DNA:

(Amount correspondent to 80 wells = 16 species)

Ø  Put 10.0 µl of 1 mg/ml photobiotin solution into prepared probe DNA in the dark room.

Ø  Irradiate with a sunlamp for 15 mins in the ice bath. (open the cover of eppendroff).

Ø  Mix well every 5 min. (by tapping).

Ø  Add 0.2 ml of 0.1 M Tris-HCl Buffer (pH 9.0) & vortex.

Ø  Add 0.1 ml of 1-butanol & vortex.

Ø  Spin for 10-20 Sec.

Ø  Discard the upper layer (Upper layer pink Solution).

Ø  Add 0.1 ml of 1-butanol & vortex.

Ø  Spin for 10-20 Sec.

Ø  Discard the upper layer (remove the unused photoprobe biotine).

Ø  Boil for 15 min.

Ø  Immediately cool on ice (quickly).

·               Dissolve in 10 ml of hybridization solution (to be 0.5 µg/ml-2 µg/ml).

·               Discard the prehybridization solution from the microplate wells.

·               Dispense 0.1 ml of biotinylated DNA solution mentioned above into microplate wells.

·               Seal the microplate wells.

·               Incubate for 2-3 hrs. at hybrid temperature or overnight.

    (Hybrid temp. : 0.41 X (G+C mol% probe DNA) + 24.3⁰C overnight is better, for we could do the        high  fluoresce intensity.

3.     Detection:

Ø  Prepare the following Solution just before experiment:

a.      Solution-1

b.      Solution-2

c.       4-MUF-Gal Solution

Ø  Discard hybridization solution and wash the wells 3 times using 0.2 ml 2X SSC.

        Enzyme adhesion to biotin-labeled DNA:

Ø  Add 0.2 ml of Solution-1

Ø  Incubate at room temp. for 10 min.

Ø  Discard the Solution-1

Ø  Add 0.1 ml of Solution-2.

Ø  Incubate at 37⁰C for 30 min.

·                  Discard solution-2 and wash each wells three times using 0.2 ml of 1X PBS.

·                 Add 0.1 ml 4-MUF-Gal Solution quickly  (in order of the wells).

·                Measure fluorescence with microplate reader every 15 mins. Keeping it to incubate at 37⁰C.  

      360/40 , 460/40

              Ø  Check the fluorescent intensity value not over 10,000.

Fig: 96 trans-well plate for DDH

            Reagents for DDH

          1.     Probe DNA (Sample DNA):

Ø  Adjust the Concentration to 1000 in nanodrop  reading (during Abs. measurement)

2.     Target DNA (Reference DNA):

Ø   Adjust the Concentration to 1000 in nanodrop  reading (during Abs. measurement)

3.     Salmon Sperm DNA (For negative control):

Ø  Adjust the Concentration to 1000 in nanodrop  reading (during Abs. measurement)

4.     PBS (Phosphate Buffer Saline) Buffer :

Ø  No need to prepare, available in lab.

Ø  20 x PBS buffer available in lab.

Ø  Dilute to required concentration

                  For 1 X PBS (For 100 ml):

·         5 ml 20 x PBS buffer

·         95 ml 3d/w water

·         Keep in room temperature.

                  For 2 X PBS (For 100 ml):

·         10ml 20 x PBS buffer

·         90 ml 3d/w water

·         Keep in room temperature

5. PBSM buffer:

Ø  500 ml 2 X PBS

Ø  300 ml water

Ø  100 ml MgCl₂ (1M)

            For 90 ml PBSM buffer:

·         50 ml 2 X PBS

·         30 ml water

·         10 ml MgCl₂

6.     1 M MgCl₂ (for 100 ml):

                 (Usually, MgCl₂.6H₂O is available in lab, So, mol. Wt. = 203.30)

Ø  20.33 g MgCl₂.6H₂O

Ø  100 ml 3 d/w

Ø  Autoclave

7.     Denatured Salmon Sperm DNA (For making prehybridisation solution) (100 µg/ml ) ( for 10 ml):

Ø  Take 0.1 ml of Salmon Sperm DNA (from Stock of 10 mg/ ml Concentration)

Ø   Take 9.9 ml µl of 3 D/w (Sterile)

Ø  Boil for 10 min. & immediately cool in ice.

10.     Hybridization Solution:

                  (Prehybridization Solution & 5% Dextran sulfate)

Ø  5 g Dextran Sulfate (5%)

Ø  100 ml prehybridization Solution

Ø  Dissolve Carefully

Ø  Store in -20⁰C

11.      Photobiotin Solution (1mg/ml)

12.      0.1 M Tris-HCL Buffer (pH 9.0) (For 100 ml):

Ø  1.21 g Tris

Ø  100 ml  Sterile 3D/w

Ø  Adjust pH to 9.0

Ø  Store in room temp.

13. 1- Butanol

14.  SSC (Saline Sodium Citrate) Buffer (For 20x Concentration):

Ø  20x 0.15 M Nacl                                 = 175.32 g

Ø  20x 0.015 M Sodium Citrate         = 88.23 g

(Sodium Citrate.H20)

Ø  3D/w                                                     = 1000ml

Ø  Autoclave

                  

                    For 2x SSC (for 100 ml):

Ø  Take 10 ml 20x SSC and dissolve in 90 ml Sterile 3D/w.

15. Solution -1 (For 100 ml):

                  (0.5% BSA, 0.1% Triton X-100, 1x PBS)

Ø  BSA (Bovine Serum albumin)      = 0.50 g

Ø  Triton X-100                                        = 100 µl

Ø  1x PBS                                                   = 100ml

16. Solution-2 (for 10 ml):

                  (Solution-1 & 10ng/ml Streptavidine-β-galactosidase)

Ø  Streptavidine- β-galactosidase   =10 µl

Ø  Solution-1                                           = 10ml

17.  4-MUF-Gal solution (4-Methylumbelliferyl β-D-galactopyranoside):

Ø  1M MgCl₂                                                                   = 10 µl

Ø  4-Methylumbelliferyl β-D-galactopyranoside      = 1 mg

Ø  1x PBS                                                                       = 10 ml

Ø  N,N-dimethyl formamide                                         = 100 µl

                 Note: 1 mg 4-MUF-Gal  is suspended in 100 µl of N,N-dimethyl formamide &                              sonicated for 3 min.

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