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GUS & LUC BOMBARDMENT ASSAY
1. Bombarded tissue is incubated according to the experiment
2. grind to a fine powdcr with a mortar and pestle in lN2
3. add 500u1 Extract Buffer l00nmM K2HPO4/kH2P04, pH 7.8 1mMDTT. Keep on ice until spin 10' 4C in microfuge. Aliquot about 450ul to a fresh tube. Freeze in lN2 and store -80C.
Mix pr assay 125u1 2 x MUG amd 50u1 100% Methanol
prewarm at 37C and at t0 add 75ul extract, mix.
When all have been done (up to 50) take out 50u1 aliquots to 250u1 0.3M Na2CO3 prealiquoted into fluoroscan microtiter plates (Microstrips black Ps, cat no. 9502177, Lab systems). These are measured in scanner (Flurorscan II, Labsystems, Finland) and the numbers are t0 values. Remember to include proper - control without bombardment for background (Tback0). Depending upon the levels of activity, generally incubate the rest for 24hr although you can see a slight color change in 2-4 hr if levels are high. Calculate t24-T0 minus Tback24-Tback0. If the values are above 5000, make dilutions as measurements are not linear at this level.
Extract buffer: good for both GUS and LUC measurements
275ml O.2M K2HP04 plus ca. 20ml 0.2M KH2PO4, adjust to pH 7.8, then add 1 vol H20.
Autoclave. Just befrore use add DTT to lmM and leupeptin to 20ug/ml.
2 x MUG: 2mM MUG in 5OmM Na3PO4/Na2HPO4 pH 7, 10mM EDTA, 0.1% Triton X-100, 0.1% Sodium Lauryl Sarkosyl, lOmM DTT. Store in aliquots at -2OC.
Luminometric LUC assay:
20ul lO x (12.5x) LUC buffer
10ul 100mM ATP
1ul 100mg/ml BSA
total is 200ul/special tube for luminometer
2. add 50ul extract, mix
3. Inject 100ul diluted luciferin solution and take measurement after 5-15". This time interval must be constant for all samples!
10 x LUC buffer
250mM Tricine, pH 7.8
in H20 adjust to pH 7.0, sterile filter, store aliquots at -20C
lOmM Luciferin stock:
D(-) Luciferin lOmg (cat 411400 (Boehringer). For 10mM stock, weigh out 1mg (light sensitive!), add 27ul DMSO to solubilize, then add 12ul 3M NaOAc, pH5, mix, then 275ul H2O, Don't make up large amountrs as this is not very stable, even at -80C.
Diluted luciferin solution: 0.5mM in H2O