Myeloid Leukemia

74 Branford and Hughes
14. Ampicillin: 25 mg/mL in sterile water. Filter sterilize through a 0.22-µm filter.
15. LB-ampicillin agar plates: Add 4 g of bacto-agar to 200 mL Luria-Bertani (LB)
medium, autoclave, and cool to 55°C. Add 0.3 mL of ampicillin and pour into
Petri dishes. Store the plates at 4°C after allowing to set.
16. 0.1 M isopropyl-β-thio-galactoside (IPTG). Filter sterilize through a 0.22-µm filter.
17. 50 mg/mL X-gal (Promega).
18. Plasmid midi kit (Qiagen). Store at room temperature.
19. Sca1 restriction enzyme (New England Biolabs).
20. Plasmid diluent: 1 mM Tris-HCl (pH 8.0), 0.1 mM ethylenediamine tetraacetic
acid (EDTA), and 50 µg/mL salmon sperm DNA. Store at 4°C.
2.4. Quality Control Samples
1. Trizol RNA isolation solution (Invitrogen).
2. RNAse- and DNAse-free 2.0-mL microcentrifuge tubes (Quantum Scientific).
3. BCR-ABL-positive cell lines.
4. BCR-ABL-negative cell line.
5. Tissue-culture media and reagents—RPMI-1640, Dulbecco’s modified Eagle’s
medium (DMEM), phosphate-buffered saline, trypsin, L-glutamine, penicillin/
streptomycin, fetal calf serum (CSL).
6. Tissue-culture flasks (Cellstar).
2.5. Real-Time Quantitative RT-PCR
1. Primer Express software (Applied Biosystems).
2. TaqMan fluorescent hybridization probes (Applied Biosystems). Probes are prepared
with a 5� FAM reporter dye and a 3� 6-carboxytetramethylrhodamine
(TAMRA) quencher dye. Reconstitute the lyophilized material in the appropriate
amount of sterile water to give a concentration of 10 µM. The probe is supplied
with a pmol concentration; therefore, divide the pmol value by 10 to calculate the
volume to add to the probe. Store in 30-µL lots at –20°C. Do not freeze and thaw
more than five times.
3. PCR primers. All primers are ordered as a 50 µM solution and stored in 30-µL
lots at –20°C.
4. TaqMan Universal PCR Master Mix (Applied Biosystems).
5. MicroAmp Optical 96-well Reaction Plate (Applied Biosystems).
6. MicroAmp Optical Cap (Applied Biosystems).
7. ABI Prism 7700 Sequence Detection System (Applied Biosystems).
8. ABI Prism SDS software (Applied Biosystems).
9. QC Reporter 2.0 software program (Chiron Healthcare).
2.6. Qualitative PCR for Atypical BCR-ABL Transcripts
1. PCR primers. All primers are ordered as a 50 µM solution and stored in 30-µL
lots at –20°C.
2. 10 mM stock of dNTPs (Amersham Biosciences). Store at –20°C in 100-µL lots.
3. 25 mM MgCl 2 (Applied Biosystems). Store at –20°C.