plant surface microbiology.pdf

29 Functional Genomic Approaches for Studies of Mycorrhizal Symbiosis 577
12. The ratio of white (recombinant) to blue (nonrecombinant) plaques will
give a quick estimate of recombination efficiency. A successful ligation
will result in at least 80 % recombinants.
13. Single plaques are isolated in 500 ml of SM buffer+20 ml of chloroform and
stored at 4 °C.
Note: lTriplEx2 phages from isolated single plaques can be converted into
pTriplEx2 plasmids by in vivo excision using the E. coli strain BM25.8 and following
the manufacturer’s instructions (Clontech, CA, USA). A brief protocol is
given below.
4 Conversion Protocol
1. Pick a single, isolated colony from the working stock plate of BM25.8 host
cells (prepared similarly to XL-blue cells as described in phage titration
protocol above) and use it to inoculate 10 ml of LB broth in a 50-ml test
tube or Erlenmeyer flask. Incubate at 31 °C overnight with shaking (at
150 rpm) until the OD 600 of the culture reaches 1.1–1.4.
2. Add 100 ml of 1 M MgCl 2 to the 10-ml overnight culture of BM25.8 (10 mM
final concentration of MgCl 2).
3. Pick each well-isolated plaque from the titration plates and place it in
350 ml of phage buffer in 96-well plates. Mix the contents thoroughly using
an 8 or 12 channel pipette and allow phage to elute at 4 °C overnight.
4. In a deep 96-well plate combine 100 ml of overnight cell culture with 100 ml
of the eluted plaque from each well. (Save the remainder of the eluted
plaques in case you need to repeat the conversion.)
5. Incubate at 31 °C for 30 min without shaking.
6. Add 200 ml of LB broth and cover the plate with the lid provided.
7. Incubate at 31 °C for an additional 1 h with shaking (225 rpm).
8. Using a multichannel pipette transfer 1–5 ml of infected cell suspension
into a 96-well LB/carbenicillin plate to obtain colonies and grow at 31 °C.
9. Pick bacterial growth from each clone and prepare plasmid DNA. For high
throughput processing use Qiagen (Qiagen, CA, USA) or Eppendorf
(Eppendorf, MA, USA) 96 format plasmid Miniprep kits. The isolated plasmid
DNA should be pure enough for direct sequencing. The pTriplEx2
sequencing primers provided may be used with standard ds-DNAsequencing
protocols.
4.1 Evaluation of the Quality of the cDNA Library
To check the quality of the cDNA library two factors must be considered: (1) the
number of primary recombinants (at least 10 5 –10 6 ) and (2) the insert length.
Insert size can be estimated by PCR with primers flanking the insertion site.