A PCR based method was used to prepare DIG-labelled probes. The 25 μl PCR mixture contained 1×PCR buffer; 1.5 mM MgSO4, 0.4 μM of each primer and 0.02 unit Taq DNA polymerase enzyme (Invitrogen). The dNTPs used for DIG-labelling PCR contains 0.05 mM of each dATP, dCTP, dGTP and 0.033 mM dTTP and 0.016 mM Digoxigenin-11-2’-deoxy-uridine-5’-triphosphate, alkali-labile (DIG-11-dUTP, Roche). 1 μl of the above dNTP mix was added to 25 μl PCR reaction mixture. The PCR conditions were the same as the standard PCR programme (section 2.3.2).
2.5.2 Probe concentration determination
To determine the labelled probe concentration, serial dilutions of probe were made (1/5, 1/25, 1/125, 1/625, 1/3125 and 1/15625). 2 μl of each dilution was spotted on a Hybond-N+ membrane (Amersham). The DNA was fixed to membrane using a UV crosslinker (Ultralum). The hybridization and detection procedure was as described in section 2.5.5 and 2.5.6. Figure 2.1 shows an example of probe concentration detection. The 1/3125 dilution spot can be clearly observed, because the incorporation of probes to the DNA template was not 100%, the actual working probe concentration used was one grade higher than detected probe concentration, which is 1/625 dilution. In the experiment, if the hybridization buffer used was 30 ml, then 48 μl of undiluted probe was added to the hybridization buffer. (30 ml /625= 48 μl)
Figure 2.1 An example of probe concentration detection
1/5 1/25 1/125 1/625 1/3125 1/15625
2.5.3 Southern blot
2 μg of each restriction endonuclease digested genomic DNA were separated on a 0.8% agarose gel at 40 V overnight at 4°C (section 2.2.5 and 2.2.7). The gel was stained in ethidium bromide for 20 minutes, visualized and photographed using Gel Documentation system (BioRad) and Quantity One 4.4.0 basic software with a ruler alongside. The gel was treated with 0.25 M HCL for 15 minutes for depurination when it contained DNA fragments bigger than 5 kb. Then the gel was incubated in denaturation buffer (A2.11) for 45 minutes followed by neutralization buffer (A2.11) for 1 hour. Finally the gel was incubated in 20 x SSC solution (A2.11) for 20 minutes. A blotting apparatus was constructed as outlined in the DIG application manual for filter hybridization (Roche). Then the gel was transferred to the blotting apparatus to allow transfer of digested genomic DNA to the Hybond-N+ membrane (Amersham) overnight. The membrane was washed in 2 x SSC for 1 minute and fixed under the UV crosslinker (Ultralum). The crosslinked membrane was allowed to air dry and was ready for hybridization.
2.5.4 Hybridisation of DIG labelled probe
The membranes were prehybridised in DIG Easy Hyb solution (Roche, approximately 10 ml/100 cm2 of membrane) at 42°C for 2 hours in a hybridization tube with rotation. The probe was incubated for 10 minutes in a boiling water bath and immediately chilled on ice to denature it. The denatured probe was added to the hybridisation buffer and incubated at 42°C overnight with rotation. Because the hybridization has 50% formamide, the hybridization buffer containing the denatured probe can be re-used following denaturation at 68°C water bath for 10 minutes. After overnight incubation, the membrane was washed 2 x 5 minutes in washing solution Ι (A2.11) at room temperature, then 2 x 15 minutes in washing solution ΙΙ (A2.11) at 68°C.
2.5.5 Immunological detection
To detect the hybridisation bands, the membrane was rinsed in buffer Ι (A2.11) (Roche) then incubated in buffer ΙΙ (buffer Ι + 1% blocking reagent) (Roche) for 30
minutes. The anti-DIG-Ap conjugate, 1:10000 dilution (Roche) was added to buffer ΙΙ and incubated for another 30 minutes. Then the membrane was washed 2×15 minutes in buffer Ι and equilibrated in buffer ΙΙΙ (A2.11) (Roche) for 5 minutes. The membrane was placed in an A4 copysafe pocket and CSPD lumigen (Roche) was added to membrane directly. The membrane was incubated at 37°C for 10 minutes to activate the enzyme, then it was exposed to x-ray film (Fuji film) for 20 minutes to 1 hour. Finally the x-ray film was developed using a 100 plus Automatic x-ray Processor (All Pro imaging).
2.5.6 Stripping blot
To re-hybridise the membrane with another probe, the membrane was washed in MilliQ water for 1 minute and incubated in stripping buffer (A2.11) at 37°C for 2 x 20 minutes.
2.5.7Screen the library for positive clones 2.5.7.1 Colony lifts
50 μl transformation culture from 1 ml of a ligation for a sub-genomic library was plated on a LB + ampicillin plate. Duplicates were made of the primary plates and stored at 4°C. A Hybond-N+ membrane (Amersham) was placed on one primary plate for 1 to 2 minutes to lift the colonies, the membrane was marked to provide orientation with the same way as the primary plate. The membranes contain colonies that were grown face up on LB + ampicillin plates for 6 hours and then grown on LB + ampicillin + 250 μg/ml chloramphenicol plates at 37°C overnight. The high concentration of chloramphenicol will stop the cells growth but allow the plasmids to increase in copy number.
After incubation, the membranes were placed face up on filter paper containing denaturation buffer (A2.11) for 15 minutes to lyse the cell membranes. Then incubated with filter paper containing neutralization buffer (A2.11) for 15 minutes and 2 x SSC solution for 10 minutes. The membrane was allowed to dry and fixed under a 26
UV crosslinker. The membrane was incubated in 10 ml 2 x SSC with 2 mg/ml proteinase K solution at 37°C with shaking for 2-4 hours. Finally the membrane was washed in 2 x SSC solution and allowed to air dry and was ready for hybridization.
2.5.7.2 Screen the library for positive clones
The membranes were hybridized with one DIG-labelled probe as described in section 2.5.4. The hybridization spots were detected as described in section 2.5.5. To re-hybridise the membrane with another probe, the membrane was stripped as described in section 2.5.6.
2.6 DNA sequencing