plant surface microbiology.pdf

572
Kbp
5.1 -
2 -
1.3 -
Gopi K. Podila and Luisa Lanfranco
MW dscDNA
Fig. 1. Analysis of double stranded cDNA synthesis products.
Lane MW is molecular weight markers in kilobase
pairs. The bright smear ranging from 4–1 kb in lane
dscDNA shows a good spread of cDNA fragment sizes
3.1.3 Reparation of cDNAs for Ligation: Proteinase K Treatment and SfiI
Digestion
1. Transfer 50 ml of the ds cDNA into a new tube, add
2 ml of proteinase K (20 mg/ml)
Incubate at 45 °C for 20 min.
2. Add 50 ml of H 2O.
3. Mix contents and spin the tube briefly.
4. Incubate at 45 °C for 20 min. Spin the tube briefly.
5. Add 50 ml of deionized H 2O to the tube.
6. Add 100 ml of phenol:chloroform:isoamyl alcohol (25:24:1;v/v/v) and mix
by continuous gentle inversion for 1–2 min.
7. Centrifuge at 10,000 g for 5 min to separate the phases.
8. Remove the top (aqueous) layer to a clean 0.5-ml tube.
9. Add 100 ml of chloroform:isoamylalcohol (24:1, v/v) to the aqueous layer.
Mix by continuous gentle inversion for 1–2 min.
10. Centrifuge at 10,000 g for 5 min to separate the phases.
11. Remove the top (aqueous) layer to a clean 0.5-ml tube.
12. Add 10 ml of 3 M sodium acetate, 1.3 ml of glycogen (20 mg/ml) and 260 ml
of room-temperature 95 % ethanol. Immediately centrifuge at 10,000 g for
20 min at room temperature.
13. Carefully remove the supernatant with a pipette. Do not disturb the pellet.
14. Wash pellet with 100 ml of 80 % ethanol.
15. Air-dry the pellet (~10 min) to evaporate residual ethanol.
16. Add 79 ml of deionized H 2 O to resuspend the pellet.
Note: Proteinase K treatment is necessary to inactivate the DNA polymerase
activity.