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)
- Resuspend IAA in ethanol at a ratio of approximately 11 uL ethanol to 2 mg IAA. Vortex until dissolved. 
- Add mixture to ELGA water to achieve a final IAA concentration of 1.75 mg/mL (10mM). 
- Sterilize with 0.22 µm filter. 
- Separate into 50 uL aliquots and store at -20°C. 
Indole-3-acetamide (Auxin precursor, IAM)
- Resuspend IAM in ethanol at a ratio of approximately 20 uL ethanol to 1 mg IAM. 
- Add mixture to ELGA water to achieve a final IAM concentration of 1.75 mg/mL (10mM). 
- Sterilize with 0.22 µm filter. 
- Separate into 50 uL aliquots and store at -20°C. 
50mM O6-Benzylguanine (O6BG)
- Dissolve 12 mg of O6BG in 1ml of DMSO. 
- Sterilize with 0.22 µm filter. 
- Separate into 50 uL aliquots and store at -20°C. 
25mM Guanine
- Dissolve 7.5 mg of guanine in 2 ml of 0.2N NaOH/Water. (Guanine can not be dissolved with DMSO/Water) 
- Sterilize with 0.22 µm filter. 
- Separate into 50 uL aliquots and store at -20°C. 
50 μM AP1903/Rimiducid (1000x stock - old DSP)
- Dissolve 1 mg of AP1903 in 14.2 mL of .1 % DMSO/water. 
- Sterilize with 0.22 µm filter. 
- Separate into 50 uL aliquots and store at -20°C. 
1 mM or 100 µM AP1903/Rimiducid (100kx or 10kx stock - current NBW)
- Dissolve 5 mg bottle of AP1903 in 3.542 mL DMSO. 
- Sterilize with 0.22 µm filter. 
- Separate into roughly 7 x 500 µL 100kx aliquots and store at -20°C. 
- Further dilute each 500 µL 100kx aliquot into 1kx by adding 4.5 mL DMSO and separate into roughly 10 x 500 µL 10kx aliquots and store at -20°C. 
Based on working conc. of 10 nM to activate iCasp9 (Mammalian Genomic Manipulation with Orthogonal Bxb1 DNA Recombinase Sites for the Functional Characterization of Protein Variants, 2023)
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:
- Make 5 mM HEPES from the 1M HEPES stock: 45 µL of 1M HEPES + 9 mL sterile water 
- Add 1 mL of 10 mg/mL puro to the 9 mL of 5 mM HEPES 
- Sterilize with 0.22 μM filer 
- Split into 50 μL aliquots and store at -20°C