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Methods

 

Bremia lactucae germination and sporulation


Lettuce cultivar Lactuca sativa cv. Cobham green is a susceptible host of B. lactuca, which has been used to generate sporangia for DNA extraction (Sicard et al., 2003). Isolates£© have been collected and, single-spored and cultured on cv. Cobham Green. These existing isolates were collected in UK but more coming isolates will be collected from Euro and American.

Lettuce cv. Cobham green seeds (supplied by Toszor seeds Ltd., UK) were sown in 90mm crystallizing dishes filled with vermiculite moistened by Hewitt¡'s solution (Hewitt and Smith, 1975). Lettuce seeds were placed on double layers of moist filter paper on top of the vermiculite (Kapooria  and Tjallingii, 1969), sealed in sandwich box then cultured in growth room at 20ºC (Kapooria and Tjallingii, 1969). After 7 days culture, B. lactucae was inoculated on Cobham green seedlings. Infected leaves of Cobham green seedlings were used to infect lettuce cultivars by swabbing the cotyledons with B. lactuace sporangia. After treatment, the infected plants were moved to a pathogen culture room (18ºC). Sporulation was observed 4-5 days later, after 7 days the final density of B. lactucae conidiophores per unit of leaf surface was sufficient for DNA extraction and in re-inoculation.

 

DNA extraction


Three methods of DNA extraction were used.

 

  • Method 1: Frozen infected cotyledons were freeze dried for 3 days, and then the freeze dried tissue was ground by FastPrep FP120 for 5.5m/second for 20 seconds (Clewes et al., 2007). DNA extractions were undertaken using Qiagen DNeasy Plant Mini Kit (Qiagen Ltd., United Kingdom) according to manufacturer's protocol.

 

  • Method 2: Fresh infected lettuce leaves were placed in a 1.5 ml tube and washed using Reverse Osmosis (RO) water. The leaves were removed after washing. The suspension was centrifuged at a maximum speed for 5 minutes, and the supernatant was discarded, 400ml RO water was added before boiling in a heated block for ten minutes. Samples were left on ice prior to use or stored at -20ºC.

 

  • Method 3: Invitrogen ChargedSwitch gDNA Plant Kit. Large amount spores are required in this methods. Fresh infected lettuce leaves were placed in a flask and washed using iced Reverse Osmosis (RO) water. The suspension was centrifuged at a maximum speed for 20 minutes, and the supernatant was discarded. Added 1ml d water in the tube votexed and transfered to a 1.5ml tube, The suspension was centrifuged at a maximum speed for 1 minute, discarded supernatant, stored in -80ºC before DNA extraction. Follow Invitrogen's protocol for DNA extraction.

 

Gel Electrophoresis


PCR products were separated by using 1.5% Agarose Gel stained with 0.5x Gel Red, Spreadex gel EL 800(Elchrom) and 6% Poly(NAT) gel(Elchrom) stained with Sybr Gold. For electrophoresis, PCR product was loaded with 5x loading buffer. Samples were electrophoresed alongside DNA mass ladder (Hypeladder IV for Agarose gel (Bioline) or 100bp ladder (Invitrogen) for Spreadex gel and Poly(NAT)).
1.5% agarose gels were electrophoresed in 1X TBE buffer at 120 V for 1hour, alongside Hyperladder IV. Spreadex gel EL 800 and 6% Poly (NAT) Gel, were electrophoresed in 0.33X TAE buffer at 55ºC in SEA 2000 gel tank at 10V/cm


Cloning


Purified PCR products were cloned into pSTBlue-1 using AccepTorTM vector Kit (Novagen). To verify the presence of the insert, transformants were picked and transferred to a replica plate with required antibiotics. Following transfer, the pipette tip used to pick the sample was against in 50ul RO water, then the stick was removed and water boiled for 5 minutes, centrifugated for 1 minute at maximum speed. 5ul was used in subsequent PCR amplifications. Where the presence of an insert was confirmed by PCR, these amplicons were sequenced directly.

Below is the map of pSTBlue-1 vector more detail can be found here.

vector.jpgClick the image to enlarge.

 

 

Useful Links

 

 

elchrom

 

invitrogen

 

qiagen

 

biolab