Qiaprep spin mini kit

The gel purified PCR products (see Supplemental Fig. S1) were cloned in pGEM-T Easy vector system (Promega). The ligation reaction was incubated at 4 °C for 10 hrs, and the ligation mix was plated on LB ampicillin plates with E.coli DH5α strain as host. The plates were then incubated overnight at 37 °C. The positive clones were selected by picking a single colony, grown in 5 ml LB Broth with ampicillin antibiotic (100 µg/ml), and incubated overnight at 37 °C. Plasmid DNA was isolated from the culture by QIAprep Spin Mini Kit (Qiagen). The positive clones were screened by restriction digestion of plasmid DNA with EcoRI in a 10 μl reaction volume at 37 °C for 2 hrs. The digested products were analyzed on a 1.5% agarose gel after electrophoresis and the amplified bands were screened for positive clones by restriction digestion of the products with EcoR1 (see Supplemental Fig. S2). Three clones from each individual were subjected to sequencing from LabIndia and SciGenom laboratories by dideoxy chain termination method. CCR5 open reading frame (ORF) was then translated to amino acids by Gene Runner and the amino acid sequences were aligned with reference sequence (NM_000579) by ClustalW to identify novel mutants.

The gel purified PCR products were cloned in pGEM-T Easy vector (Promega). The ligation reaction was incubated at 4°C for 10 hours then the ligation mix was added to LB ampicillin plates with E.coli DH5α strain. The plates were incubated overnight at 37°C. The positive clones were selected by picking a single colony and grown in 5 ml LB Broth with ampicillin (100 µg/ml) and incubated overnight at 37°C. Plasmid DNA was isolated from the culture by QIAprep Spin Mini Kit (Qiagen). The positive clones were screened by restriction digestion of plasmid DNA with EcoRI in a 10 µL reaction volume at 37°C for 2 hours. The digested products were analyzed on a 1.5% agarose gel. The positive clones were sequenced from LabIndia and SciGenom laboratories.

To verify ITD mutation, FLT3 DNA fragments were excised for DNA purification. The DNA was purified by using a Qiaex II gel purification kit (Qiagen, Valencia, CA) according to the manufacturer's protocol. The purified DNA fragments were subcloned into pTA2 vectors using a TA cloning kit (Target Clone 0811, Toyobo, Japan) and were subsequently transformed into competent Escherichia coli cells according to the manufacturer's instructions. White colonies were isolated and cultured in lysogeny broth. Plasmids were purified using a QIAprep Spin Mini Kit (QIAgen, Crawley, West Sussex, UK) and sequenced in both directions using T7 and M13 primers.