Production of a a phage lambda library of genomic DNA

Genomic DNA isolation

 

DNA was precipitated by addition of 500 µl 100% ice-cold ethanol and the mix was homogenized by inversion for 1 to 3 mins, centrifuged for 2 mins at 4000g at 4°C and the supernatant was discarded. The pellet was washed with 1 ml 75% ethanol and centrifuged at 7000 rpm for 2 mins at room temperature. The pellet was eluted into 200µl milliQ water on ice bath for 10 mins.

A phenol-chloroform step was finally performed to purify the DNA. An equal volume of phenol-chloroform was added and the content of the tube was mixed by gentle inversion until the pellet was dissolved. The tube was centrifuged at 10 000 rpm for 10 mins at room temperature. The upper aqueous layer was transferred to a new tube. To eliminate any residual phenol, an equal volume of chloroform was added to this phase and the mix was centrifuged at 10000 rpm for 5 mins at room temperature. The supernatant was collected for the rest of the protocol. The phenol phase was kept as we observed some remaining DNA at the white layer that remains on the surface. This remaining DNA was eluted from the phenol layer by addition of 200 µl milliQ water and phenol-chloroform extracted as explained above.

The quantity of DNA was evaluated by spectrophotometer (approximately 2.5 mg in a final volume of 400 µl). 5 µl was loaded on a 0.7 % agarose gel to evaluate integrity by visual inspection as illustrated in figure 1.

 

Digestion and fill-in

 

Partial digestion:

 

Table 1: Partial digestion at restrict scale of enzyme concentration. The quantity are given in μl

 

 

A thorough digestion was then performed in the appropriate range using increasing concentration of enzymes increased twice in between each reaction condition. Each reaction condition was performed 10 times, each time in 30 µl. The 10 fractions were pooled and an aliquot was loaded and size separated on a 0.4% gel agarose by electrophoresis. This allows to chose the condition that was optimal for generation of the 10 to 23 kb bands. An example of such a digestion on a gDNA preparation at 6.5 μg/μl is illustrated on figure 3 and reaction mixes were prepared as outlined in table 2. In this example, sample 2 was chosen for further step.

 

 

Table 2: Partial digestion at restrict scale of enzyme concentration.

 

 

 

 

Partial fill-in

Digestion reactions that produced fragments of expected size were purified doing a phenol-chloroform as previously detailed. The upper layer (approximately 300 µl) was collected into a fresh tube and used for the next steps of the protocol.

 

Table 3:

 

 

 

 

 

Fig. 4: Partial fill-in of gDNA with the Klenow Fill-In kit (Stratagene, La Jolla, CA). The reaction was performed as outlined in Table 3. Migration was performed using 1µl of filled-in sample and 1 µg of pUC19 DNA on a 0.7% agarose gel in TBE at 100V for 2h at room temperature. (1) plasmid control, (2) filled in sample.

Ligation into the lambda vector

 

 

Table 6: Reaction mix for ligation and self-ligation (control)

 

Packaging

 

 

Bacterial cultures for transformation by the recombinant phage, titration and amplification of the library.

 

Bacterial culture:

 

Titration of the library:

4.5 ml of pre-warmed (48°C) LB Top agar was added to the transformed bacterial strain and immediately poured on the pre-warmed (37°C) LB agar plates. The plates were cooled at room temperature for several minutes and incubated upside down at 37°C for 15 h. Phage forming units were counted after overnight incubation. Plates can be stored at 4°C for several weeks.

 

Genome coverage estimation:

(Total number of pfu * mean size of insert (15kb)) / (genome size)

 

Amplification of the library:

This amplified library was titled as previously described.

 

 

Synthesis of the probes:

 

Table7: PCR conditions for probe amplification

 

 

 

Performing plaque lift (Transfer to membranes):

 

Estimation of optimal hybridization temperature:

 

For UR1:

 

Prehybridization:

The ref for the roller tubes.

 

Hybridization:

4.4 µl of DIG-labeled probe (1 µg/µl) was used for each membrane. The probe was denatured during 5 min at 99°C and was immediately cooled on ice. The denatured probe was added to 20 ml of DIG easy hyb and poured into each roller tube. Incubation was performed overnight (16-19h) with rotation in a hybridization oven at 42°C.

 

Stringency washes:

After overnight incubation, DIG easy hyb buffer containing the probe was removed from the roller tubes  and washes were performed in the same roller tubes in the hybridization oven.  First, two low stringency washes were performed with 200 ml washing solution 1 (2X SSC, 0.1% SDS) for 5 min. Then, two high stringency washes were performed in 200 ml of washing solution 2 (0.5X SSC, 0.1% SDS) for 15 mins at 68°C. Finally, two very high stringency washes were performed in 200 ml of solution 3 (0.2X SSC, 0.1% SDS) for 15 mins at 68°C.

 

Chemiluminescent detection:

The next steps were performed at room temperature in the roller tubes using the hybridization oven. After hybridization and stringency washes, the membrane was rinsed once in 200 ml of washing buffer for 5 min at room temperature. Then, washing buffer was removed and the membrane was incubated for 30 min in 100 ml of blocking solution. The blocking solution was replaced by antibody solution for 30 min. Two washes were then performed in 200 ml of washing buffer during 15 min. The membrane was finally equilibrated during 5 min with 100 ml detection buffer.

Film exposure was performed during 1 to 3 min depending on the signal intensity.

 

Secondary and tertiary screening:

For secondary screening, the density of phage recommended is:

For tertiary screening the density of phage recommended is:

The Petri dish are then processed for UR1 detection as previously described.