=============================== Small molecule stocks for TC =============================== Antibiotic concentrations ----------------------------------------- Reference: `Selection antibiotics `_ (ThermoFisher) =============================================== ================= ========================= ============================= ========================================= Name Temperature Stock conc Common working conc. 293T conc. KL 2023.12.11 =============================================== ================= ========================= ============================= ========================================= Blasticidin -20°C 10 mg/mL (1kx) 1 - 20 µg/mL 10 µg/mL (1:1000) by 4-5 dpt Geneticin (G-418) -20°C ? mg/mL (1kx) 200 - 500 µg/mL 400 µg/mL (1:100) - no death observed Hygromycin -20°C 50 mg/mL (250x) 200 - 500 µg/mL 200 µg/mL (1:250) by 6-8 dpt Puromycin -20°C 1-10 mg/mL (1-10kx) 0.1 - 10 µg/mL 1 µg/mL (1:10,000) by 3-4 dpt Zeocin -20°C 100 mg/mL (100x) 50 - 400 µg/mL 1,000 µg/mL (1:100) >8 dpt =============================================== ================= ========================= ============================= ========================================= Other stock and working concentrations ----------------------------------------- =============================================== ================= ========================= ============================= Name Temperature Stock conc Common working conc. =============================================== ================= ========================= ============================= Doxycycline -20°C 1 mg/mL (1kx) 1 ng/mL - 1 µg/mL Grazoprevir (GZV) -80°C 1 mM (1kx) 1 nM - 1 µM Polybrene (Hexadimethrine bromide) -20°C 5 mg/mL (1kx) 5 - 8 µg/mL =============================================== ================= ========================= ============================= Indole-3-acetic acid (Auxin, IAA) --------------------------------- 1. Resuspend IAA in ethanol at a ratio of approximately 11 uL ethanol to 2 mg IAA. Vortex until dissolved. 2. Add mixture to ELGA water to achieve a final IAA concentration of 1.75 mg/mL (10mM). 3. Sterilize with 0.22 µm filter. 4. Separate into 50 uL aliquots and store at -20°C. Indole-3-acetamide (Auxin precursor, IAM) ----------------------------------------- 1. Resuspend IAM in ethanol at a ratio of approximately 20 uL ethanol to 1 mg IAM. 2. Add mixture to ELGA water to achieve a final IAM concentration of 1.75 mg/mL (10mM). 3. Sterilize with 0.22 µm filter. 4. Separate into 50 uL aliquots and store at -20°C. .. _O6-BG_stock: 50mM O6-Benzylguanine (O6BG) ----------------------------------------- 1. Dissolve 12 mg of O6BG in 1ml of DMSO. 2. Sterilize with 0.22 µm filter. 3. Separate into 50 uL aliquots and store at -20°C. .. _guanine_stock: 25mM Guanine ----------------------------------------- 1. Dissolve 7.5 mg of guanine in 2 ml of 0.2N NaOH/Water. (Guanine can not be dissolved with DMSO/Water) 2. Sterilize with 0.22 µm filter. 3. Separate into 50 uL aliquots and store at -20°C. 50 μM AP1903/Rimiducid (1000x stock) ----------------------------------------- 1. Dissolve 1 mg of AP1903 in 14.2 mL of .1 % DMSO/water. 2. Sterilize with 0.22 µm filter. 3. Separate into 50 uL aliquots and store at -20°C. Puromycin, 1 mg / mL (1,000x stock) ------------------------------------- We use dissolved `10 mg/mL puromycin `_ (Invivogen, cat. ant-pr-1). It comes as 10 tubes x 1 ml @ 10 mg/mL dissolved in HEPES buffer (5 mM, pH 7.3). To dissolve a single 1 mL @ 10 mg/mL tube: 1. Make 5 mM HEPES from the 1M HEPES stock: 45 µL of 1M HEPES + 9 mL sterile water 2. Add 1 mL of 10 mg/mL puro to the 9 mL of 5 mM HEPES 3. Sterilize with 0.22 μM filer 4. Split into 50 μL aliquots and store at -20°C