1Kb

1. 1Kb cutC cutA orf2 orf3 orf4 cutB orf5-1 cutR cutC cutA orf2 orf3 orf4 orf6 orf1 cutB orf5-3 5 : Primer 5F and 6R 1 : Primer 1F and BR 1364 1433 4 : Primer 4F and 5R 366 bp 1512 1560 3: Primer 3F and 4R 413 bp 2 : Primer 3F and 5R Primer 1F : orf1-cutB RT-F Primer BR : orf1-cutB RT-R Primer 3F : orf3del-F Primer 4R : orf4del-R Primer 4F : orf4del-F Primer 5R : orf5del-R Primer 5F : copy2-P3 Primer 6R : copy2-P2 RT-PCR cDNA(O) RT-PCR cDNA(X) PCR SM 1 2 3 4 5 1 2 3 4 5 1 2 3 4 5 1 : Primer 1F and BR : 61 ℃ 2 : Primer 3F and 5R : 61 ℃ 3 : Primer 3F and 4R : 61 ℃ 4 : Primer 4F and 5R : 61 ℃ 5 : Primer 5F and 6R : 61℃

2. RT-PCR protocol • Prepare 5 ㎍ of RNA and add DNase I (50U) in 8 ㎕ of RNase-free water and add 2 ㎕ of 5X Superscript II buffer (invitrogen) and incubate at 37℃ for 1h. • Add 1 ㎕ of primer (0.5 pmol/㎕ ) and heat 70 ℃ for 10 min. • Cool on ice. • Add 4 ㎕ of 10 mM dNTP, 1 ㎕ of 0.1M DTT, 2 ㎕ of 5X Superscript II buffer, 2 ㎕ of RNase-free water and 1 ㎕ of reverse transcriptase (200U). • Incubate at 42 ℃ for 1 h. • Heat the tube 90 ℃ for 10 min. • Go to RT-PCR. A 2 3 4 A 2 3 4