plant surface microbiology.pdf

584
Gopi K. Podila and Luisa Lanfranco
2. Include a group of negative and positive control clones with the other
clones to be printed onto the membrane.
3. Denature the PCR products in 5¥ denaturation solution (2 N NaOH, 50 mM
EDTA) diluted to 1x final concentration at 37 °C for 30 min (Jordan 1998).
4. Presoak Hybond N+ nylon membrane filters (Amersham Pharmacia
Biotech, Piscataway, NJ, USA) in 0.1 M NaOH for 1 min, then place on 3-mm
filter paper.
Note: When using a manual arrayer, one layer of filter paper on top of a mouse
pad seems to be an optimal surface for printing. Spotting can be done with a
384-pin dot-blot tool (V&P Scientific, San Diego, CA, USA) with 0.9–0.5 mm
diameter flat tip pins. If you are going to print many copies, use a multi-print
replication device (V&P Scientific, San Diego, CA, USA) to give consistent
alignment between membranes and to allow spacing for printing up to 4x384
spots on the same membrane (Schummer et al. 1997).
5. Dip the pins into the 384-well plate containing the denatured DNA. The
pins will deliver ~50 nl of sample yielding spots consisting of approximately
5 ng (the linearity of delivery can be tested by using different concentrations
of PCR products in the same volume).
6. Cross-link membranes for 30 s in UV-crosslinker at optimal setting (Fisher
Scientific, Pittsburgh, PA).
7. Neutralize the array for 5 min in a solution of 0.5 M Tris pH 7.8, 1.5 M NaCl
followed by rinsing in ddH 2O for 5 min.
8. Air dry the arrays on filter paper and wrap in Saran wrap until use.
7.4 Generation of Exponential cDNA Probes from RNA for Macroarrays
and Hybridization Analysis
We found the protocols described by Gonzalez et al. (1998) work very well.We
use components from SMART cDNA synthesis kit (Clontech, CA, USA) for
this purpose.
1. Assemble the following in a 0.2-ml PCR tube
0.5–1 mg RNA 1 ml
10 mM oligo dT primer (CDS from SMART cDNA kit) 1 ml
10 mM of SMART IV oligonucleotide 1 ml
ddH 2O 2ml
Heat the mixture to 70 °C for 2 min, spin briefly and cool at room temperature
to anneal the primers.
2. Add
5x Reverse transcription buffer 2 ml
20 mM DTT 1 ml
10 mM dNTPs 1 ml
Powerscript Reverse Transcriptase 200 U (Clontech, USA) 1 ml
Total volume 10 ml