BioRad CFX96 Real-Time PCRProtein Melt Protocol

1. Use plates provided by MSKC (BIO-RAD, Hard-Shell PCR Plates 96-well, thin-wall, Cat#: HSP9665).

2. Add all components (e.g., protein, ligand, buffer) to wells; total 50 µL in buffer of choice. For initial run (e.g., apo-protein), determine optimal concentration using 2-fold dilution series:

Well 1 2 3 4 5 6Protein Conc (mg/ml) 1 0.5 0.2

50.12

0.06

0.03

3. Add 0.5 µL of fluorescent reagent (SYPRO Orange, Molecular Probes, S6651) to each well. The working stock is 1:1 with DMSO; found in the fridge.

4. Mix each well by pipetting up and down with a p200 – avoid bubbles.5. Seal with sticky plastic cover (cut to size with razor), roll with roller, wipe off fingerprints

with Kim wipe.6. Turn on machine (Biorad CFX96 RT System C1000 Touch), wait for it to boot up, then

open software on laptop (“Protein Melt I” protocol shortcut on desktop).7. Place plate in machine. Make sure you use the button on the front of the lid to open and

close the lid (do not force it down).8. Work through the three tabs in the software:

a. Protocol – “Protein Melt I”, check it does 4oC for 5 min 4oC for 1 min 4 to 100oC, 1o degree per minute.

b. Plate - Create new, sample type “unknown”, check “load FRET” box, save plate layout.

c. Start run - Suggests file – change “admin” to “your name”.

Thanks to Shannon Sirk for providing the first document.

Important disclaimer: This example protocol is in no way meant to be optimized for your particular protein or application. This document is merely provided, as a basic guide, to get you up and running with the instrument. Suggestions/comments/additions are highly welcomed.

Please, do not hesitate to contact the MSKC staff for any enquire about the CFX.

Remember to sign up the paper form!

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