Incidence, Distribution and Characteristics of Major Tomato Leaf ...

Incidence, distribution and characteristics of major tomato leaf curl and mosaic virus diseases
Clones were spread on LB medium with Ampicillin and X-Gal coating, in plastic
petridishes. Inoculated plates were incubated overnight at 37 ºC (Maniatis et al., 1992;
Nakhla et al., 1993). Clones could grow on media with antibiotics because of pCR 2.1
TOPO® vector plasmid (Promega, 1996), which has a gene called “ampRgene or bla”
resistant to ampicillin and kanamycin (Lewin, 1997).
However, by inserting the PCR linear DNA fragment in the plasmid, the gene responsible
for breaking down X-Gal (lacZgene) was destroyed. This gene is responsible for
synthesis of β-galactosidase, which digests X-Gal leading to formation of blue colonies.
Therefore, only white colonies were picked and individually subcultured in new tubes
with 2xYT liquid media, plus ampicillin at 37 ºC, overnight ³ .
Using the standard minipreps protocol used by Maxwell Laboratory (University of
Wisconsin-Madison, 1998) DNA was extracted from cloned cultures and used for
restriction enzyme digestion. Restriction enzyme digestion products were run on a gel,
1% agarose, and in TBE buffer. Where two bands similar to those formed with tomato
leaf sample isolate RL5 DNA (ToLCV-UG) were formed again, the desired band was cut
carefully from the stained agarose gel under UV light and put in a clean eppendorf tube.
Agarose gel extraction protocol (QIAEX II, 1997) was used to purify DNA from the agar.
Subsequently, restriction enzyme digestion was done in order to determine whether
transformation had been successful and the desired DNA fragment had been cloned.
Enzyme EcoR1 in Multi-Core buffer was used in a reaction mixture of 6.5 µl of disodium
water, 2 µl of miniprep DNA, 1 µl of multi-core buffer, and 0.5 µl of the enzyme
(Srivastava et al., 1995). Enzyme Spe 1 was also used for IG1 DNA (ToLCV-UG). The
reaction mixture was incubated in a water bath for 4 hours overnight at 37 ºC. The
digestion product was run on 1% agarose gel, in TBE buffer, at 95-119 Volts. Specific
sites for enzyme digestion are as shown in Figure 3.9.
³ This cloning procedure was in use by Maxwell Laboratory, University of Wisconsin-Madison. Similar
literature can also be found in Lewin (1997) publication on GENES.
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