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.

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.

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 - old DSP)

  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.

1 mM or 100 µM AP1903/Rimiducid (100kx or 10kx stock - current NBW)

  1. Dissolve 5 mg bottle of AP1903 in 3.542 mL DMSO.

  2. Separate into roughly 7 x 500 µL 100kx aliquots and store at -20°C.

  3. 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).

Do not filter rimiducid with a PES filter! It will dissolve. You may use a PTFE filter.

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