Made by Reetika, Hagop, Shinha lab; adopted by Akiva (2016)
- Grow N.Benthamiana 2-4 weeks, preferably leaves should be fully open but not yet mature.
2.Grow agro (preferably from an up to 1 week old plate) in 10ml LB or YEP in a 50ml conical tube with 200uM of acetosyringone O.N or till O.D ~2 with antibiotics.
3.Measure O.D of the O.N agro culture, centrifuge (6K 5min) and re-suspended in infiltration media to O.D 600 of 0.1, incubate for 4hrs in a shaker at 28c (usually re-suspend in the morning and infiltrate in the evening).
- At this stage combine equal volumes of agro culture that you want to co-infiltrate. Using an agro strain with 35S-P19 should make signals last several days longer by slowing down insert inactivation.
5.Use a 1 ml syringe to infiltrate the leaves on their bottom/ abaxial side while pushing the syringe gently against your gloved finger on the other side.
- After infiltration plants should be kept in the dark for 36 hrs, whole plants can then be left for 2 more days for entrainment or leaf discs from the area around the infiltration sites can be excised (sterilized for 5 min in 10% bleach, washed 3 times in sterile water for 5 min) placed on 1xMSS plates and entrained. The plates can be put into the CEF chamber for 12:12, 16:8 L:D or any other entrainment regime.
- After entrainment spray the plates with Luciferine as specified in the LUC assay protocol and place into the CCD camera.
- 10mM MES, 10mM MgSO4 and 200uM Acetosyringone pH5.6
- Make 10mM MES, 10mM MgSO4 pH5.6 (titrate with 1M NaOH) and freeze in aliquots, add Acetosyringone before use.
Stocks:
- 100mM Acetosyringone in DMSO
- 1M MgSO4