2.3　In Vitro Synthesis of HIV-1 RNA

A plasmid pBKBH10S（kindly supplied by Dr.J.Rossi）was used as a template for HIV-1 RNA synthesis.pBKBHlOS consists of a complete HIV DNA sequence（8，927 bp）minus the LTR that is positioned downstream from a T7 RNA polymerase promoter sequence.The HIV-1 RNA standard was synthesized by in vitro transcription in a reaction containing 40 mmol/L Tris-HCl，pH 8.0，8 mmol/L MgCl2，2 mmol/L spermidine，50 mmol/L NaCl，1 μg DNA template，400 μmol/L rNTPs，25 units of RNasin and 10 units of T7 RNA polymerase.After incubation at 37℃for 30 min，the synthesized HIV-1 RNA was extracted with phenol and precipitated with ethanol.The purity and integrity of the synthetic HIV-1 RNA was determined by Northern analysis.The HIV-1 RNA was quantitated by measuring the absorbance at 260 nm.A dilution series of known copies of HIV-1 RNA was made to generate an HIV-1 RNA standard curve of copy number vs PCR product.(https://www.daowen.com)