To avoid any traces of DNA, total RNA isolated was treated with DNase (Fermentas
Cat # EN0521).
The probes (Table 1), the biotinylated universal primers (Conjugate control) and the PCR products from non-biotinylated universal primers (Amplification control), were prepared with concentrations of 2.5 pmol/[micro]d; they were then spotted onto SensiBlot[TM] Plus Nylon membranes (Fermentas
GmbFI, Germany) employing a 96-well Dot-Slot Blotter (Cleaver, UK) according to the manufacturer protocol.
RNA isolation was performed using the GeneJet purification kit (Fermentas
Hundred uL of reaction mixture was made up of 7 uL dNTPs, 5 uL of 10x PCR buffer, 3 uL MgCl2, 2 uL of each primer, 2.0 uLTaq polymerase (Fermentas
, USA), 10 uL template DNA and 10 mMTris buffer (pH 8.3) to make the volume.
Restriction digestion was done at 37[degrees]C for 5 minutes using 5 units of Fok-I fast digest enzyme (Fermentas
Next, 100 [micro]L of RCA mix [0.5 U/[micro]L Phi29 polymerase (Fermentas
) in 1X Phi29 polymerase buffer (Fermentas
) with 0.25 g/L BSA, 0.4 mmol/L dNTPs (Fermentas
), and ddH2O] was added, followed by incubation for 1-1.5 h at 37[degrees]C and then by washes.
Further RNA contamination was removed by RNase treatment (Fermentas
U.K.) for one hour at 37[degrees]C.
At specified time-points, MTB infected RAW264.7 cells were harvested and total RNA isolation was carried out using GeneJET[TM] RNA purification kit (Fermentas
Genomic DNA samples (250 ng) were digested with FastDigest EcoRI and subsequently with FastDigest Taql (Fermentas
, Hanover, MD, USA) based on the manufacturer's instructions.
The PCR products were analyzed on 1.5% agarose gel and purified by GeneJET[TM] Gel Extraction kit K0691 (Fermentas
RevertAid[TM] First Strand cDNA Synthesis Kit was from Fermentas
Lane M: 100 bp DNA ladder (Fermentas
, USA); Lane 1: negative control (IC); Lane 2: PCR amplicon of K.