by Hajiwon Yang (hajiwonyang@mail.com)
Date
26.02.2016~03.03.2016
Material
1. Culture
2. RNA Extraction
3. PCR
4. Gel Electrophoresis
Method
1. Culture
- Samples are taken from live broilers of South Korea by tracheal swab.
- Swabbed samples are suspended in 1ml of PBS in a 1.5ml E-tube.
- The samples are vortexed about 1minute.
- the samples are centrifuged at 13,000 rpm, 10 minutes.
2. RNA Extraction
- RNAs of swabbed samples are extracted by Viral Gene-spin™ Viral DNA/RNA
Extraction Kit.
- The Kit material includes Lysis buffer, Binding buffer, Wash A buffer,
Wash B buffer and Elusion buffer.
- The protocol that I followed is based on Viral Gene-spin™ Kit protocol.
(You can find that protocol easily in Internet.)
1) Transfer 150µl cell culture supernant in the E-tube.
2) Add 250µl of Lysis buffer.
3) Mix by vortexing for 15secs
4) Incubate at room temperature for 10min.
5) Add 350µl of Binding buffer, and gently vortexing.
6) Place a sping column in a provided 2ml collection tube.
7) Load lysates on the column and centrifuge at 13,000rpm for 1min.
8) Discard solution in collection tube and place the spin colmn back.
9) Add 500µl of Washing buffer A, centrifuge for 1min at 13,000rpm.
10) Same as 8)
11) Add 500µl of Washing buffer B, centrifuge for 1min at 13,000rpm.
12) Discard solution in collection tube.
13) Place the column in RNease-free E-tube.
14) Centrifuge for 2min at 13,000rpm for dry.
15) Place the column in a RNease-free 1.5ml E-tube.
16) Add 50µl of RNease-free water directly onto the membrane.
17) Incubate at room temperature for 5min.
18) Centrifuge for 2min at 13,000rpm.
3. Polymerase Chain Reaction (PCR)
- The total number of samples are 11.
: TM1, TM2, TM3, SI1, SI2, SI3, VH1, VH2, VH3, MS positive, MS negative.
- RNAs are converted into DNAs by using TaKaRa RNA PCR Kit (Ex Taq).
(∵ RNA is easily break down and weaker than DNA.)
- Converted DNAs are stored at -4ºC.
- I used the iTaq™ PCR Kit to PCR the DNAs.
- I made the "master mix" for 12 sample quantity.
(∵ possiblitiy of loss during pipetting)
(Master mix: DW → 10X buffer → DNTP → F primer → R primer → Itaq plus)
- Forward Primer: VIhA F 5'- GAT GGG TAA AAT AAA AGG AT -3'
Reverse Primer: VIhA R 3'- GCT TCT TGT TGT AGT TGC TTC -3'
- The protocol that I followed is based on Viral Gene-spin™ Kit protocol.
1) Prepare the 1.5ml E-tube, 1 PCR tube, and ice.
2) Melt the DNTP and 10X buffer. (Vortex and spin-down)
3) Make the master mix at E-tube on the ice.
(From large volume to small volume)
Add 402µl of DW.
Add 60µl of 10X buffer.
Add 48µl of DNTP.
Add 12µl of F primer and R primer.
Vortex and spin down.
Add 6µl of Itaq plus.
(Do not prearrange the enzyme. Just take off just before use.)
(∵ Enzyme can easily break down by heat)
Gently tapping with fingers to mix.
3) Separte 45µl of master mix for each PCR tube.
4) Add 5µl of each sample for each PCR tube.
5) Gently tapping with fingers and spin down.
4. Gel Electrophoresis