Ta vector pcr 2

Isolation of total RNA from MVA, conversion to cDNA, design of primer pairs, synthesis of oligonucleotides and RT-PCR were carried out as described previously (Price et al., 2014 (link)). PCR for H4L were performed with DNA from ECTV. Sequences of oligonucleotides and PCR product sizes are listed in Table 1. PCR products from E3L, 078R/G8R, 047R/F17R, and H4L were cloned into the TA-vector pCR2.1 (Invitrogen). Plasmids were linearized with the restriction enzyme SpeI and subsequently purified by phenol/chloroform extraction, precipitated with ethanol, and dissolved in RNase free water. These plasmids were then used to produce biotin-labeled RNA probes using the AmpliScribe T7-Flash Biotin-RNA Transcription Kit (Epicentre) according to the manufacturer’s instructions. To synthesize the β-actin RNA probe, cDNA from U937 cells (ECACC 85011440) was amplified using the sense primer 5’-CCT CGC CTT TGC CGA TCC-3’ and the antisense primer 5’-GGA TCT TCA TGA GGT AGT CAG TC- 3’ yielding a PCR product with a size of 626 bp (Raff et al., 1997 ). This PCR product was cloned in sense direction into pT-Adv (Clontech). The plasmid pT-Adv-β-actin was linearized with the restriction enzyme BstEII and used as template to produce a 103 bp long biotinylated β-actin antisense RNA by in vitro transcription, which was performed as previously described (Lehmann et al., 2001 (link)).