RsrII NEB#R0501

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	PCRs were purified by using a PCR cleanup kit (Qiagen) and digested with RsrII (NEB). Digested PCR products were run on a 1.8% agarose gel, stained with ethidium bromide (Sigma), and imaged by using a Chemi-Doc XRS+ system (Bio-Rad). Relative band intensities were quantified by using ImageLab 4.0 software (Bio-Rad).

Protocol tips
PCRs were purified by using a PCR cleanup kit (Qiagen) and digested with RsrII (NEB). Digested PCR products were run on a 1.8% agarose gel, stained with ethidium bromide (Sigma), and imaged by using a Chemi-Doc XRS+ system (Bio-Rad). Relative band intensities were quantified by using ImageLab 4.0 software (Bio-Rad).

Publication protocol

"C6/36 or C2C12 cells were plated into 24-well
dishes. Growth medium was removed, and triplicate wells were inoculated at an MOI of 0.01 (C6/36) or 1 (C2C12) with a 1:1, 1:10, or 10:1 ratio
of either RRV-T48 to RRV-T48 E2 Y18H-RsrII or RRV-T48-RsrII to
RRV-T48 E2 Y18H. Viruses were adsorbed onto cells for 1 h. Wells were
then washed three times with 1 ml of room temperature PBS. One milliliter of growth medium was then added to each well. Culture supernatants
were collected at 24 h postinoculation (hpi) and stored at
80°C for
analysis. Virus titers in the supernatants were quantified by plaque assays
and used to infect additional C6/36 cells or C2C12 cells at an MOI of 0.01
or 1, respectively. Total RNA was extracted from supernatants by using a
PureLink RNA minikit (Life Technologies), and cDNA was generated by
using Superscript III reverse transcriptase (Life Technologies). RRV
genomic DNA was amplified via PCR using primers designed to flank the
endogenous RsrII restriction site (forward primer, E2 9194-5=-CACTAC
CAGTACTGACAAGACC-3=; reverse primer, 6K 9869-5=-CCACAGATA
TGCCATAGTCTCAGC-3=). PCRs were purified by using a PCR cleanup
kit (Qiagen) and digested with RsrII (NEB). Digested PCR products were
run on a 1.8% agarose gel, stained with ethidium bromide (Sigma), and
imaged by using a Chemi-Doc XRS system (Bio-Rad). Relative band
intensities were quantified by using ImageLab 4.0 software (Bio-Rad)."

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