Protocols
NA preparation from plant samples by CTAB method
modified from Permingeat et al.,1998
Method was tested for Sida spec., Nicotiana spec., Abutilon, cassava, tomato, sugar beet and cotton. If you have exotic or unkown plants you may add 2mM Mercapto-EtOH and/or 1% PVPP (unsol.)
Material:
H-Buffer:
- 100 mM Tris-HCl pH 8.0
- 20 mM EDTA,
- 1.4 M NaCl,
- 2% CTAB,
- 0.5 M glucose
- 100mM DTT
CI: Chloroform:isoamyl alcohol (24:1)
Isopropanol
70% ethanol
Method:
- grind plant sample (leaf disc, 10-50mg) in lN2
- add 500µl H-Buffer and shake for 1h at 60°C
- add 500 µl CI and shake 5’
- spin down at max. speed for 5’ at 4°C
- collect upper phase and add 0.8 Vol. Isopropanol
- shake reaction tube twice, a precipitate should be visible
- spin down at max. speed for 10’ at 4°C
- wash pellet with 70% EtOH
- spin down at max. speed for 5’ at RT
- let EtOH evaporate for 5’ and dissolve pellet in 100µl H2O (store at –20°C)
if you use a FastPrep device:
- add plant sample (leaf disc) to 500µl H-Buffer
- disrupt tissue 3x for 20’’ with 4 m/s at RT
- shake for 1h at 60°C
- proceed as described above
RCA reaction
use always new tips to avoid contamination, wear gloves use 25pg of pUC19 or pBluescript as positive control.
Take water, which was used to dissolve pellet of the above performed NA preparation as negative control.
Material:
- GE Healthcare Templiphi Kit
Method:
- Add 0.5µl of NA preparation to 5µl of sample buffer
- Heat 3’ at 95°C
- Cool down on ice for 2’
- Add premix of 5µl reaction buffer and 0.2µl enzyme mix (if you have more samples prepare a mastermix)
- Incubate 16h at 30°C
Prepare a second RCA reaction, but use 0.5µl of 1:4 dilution of NA preparation Incubate 16h at 30°C
Stop reaction by heating 10’ at 65°C (store at –20°C)
RFLP
Use 2 µl of RCA product in an Hpa II restriction assay and separate fragments in a 2% agarose gel.