I have developed this one tube RT-PCR method (it took me a year of
tuning...) to identify garlic viruses from RNA preps or from dilute plant
sap (in Borate buffer). It works very well, is highly sensitive and
repetetive. This method has also been used to detect viruses using
imunocapture (substituting PBS for TBS) in plum bark sap and other
difficult starting material.
You must use AMV-RT, the buffer is incompatible with MMLV.
PBS-Tween can be substituted for TBS-Tween, or not at all
10X Sellner buffer must be autoclaved 30' to melt gelatin (put in bottle
first when dry), but b-MET must be added after. Aliqouts can be kept
frozen for a long long time so prepare a lot...
10X Sellner buffer is:
670 mM Tris-HCl,
170 mM (NH4)2SO4
10 mM beta-mercapto-ethanol
2 mg/ml gelatin (Aldrich calf skin 225 bloom)
60 uM EDTA pH 8.0
(Sellner et al, 1992).
The reaction mix is:
1. 1.5 mM MgCl2
2. 125 uM each dNTP
3. 1X Buffer Sellner
4. Triton X-100 0.03%
5. 1X PBS-Tween [8 mg/ml NaCl, 0.2 mg/ml KH2PO4 , 1.15 mg/ml Na2HPO4,
0.2 mg/ml KCl, Tween-20 0.05% ] 8%.
6. each specific primer 100 ng
7. Taq polymerase
8. AMV RT 5U (Chimerex USA is very good but very concentrated, 2.5 U
works too, but less sensitive)
9. 0.2 U Prime RNase-inhibitor (5'- 3' Inc. Boulder, USA) (optional for
total RNA, must use for direct sample aplication methods)
10. total RNA 2-5 ug
11. ddH2O to 50 ul
Glad to be of use, please let me know of the results
Yoel Shiboleth Msc
shibolet at mishkei.org.il