Team:FAFU-CHINA/Protocol
Protocol
Whole RNA Extraction
We use Trizol to extract the whole RNA of CSBV,and the detail protocol is showed as bellow:
1.Take 50-100mg tissue stored in -80℃ or liquid nitrogen. Grind the tissue into powder in mortar with liquid nitrogen. Transfer the powder into a 1.5ml EP tube(RNase-Free).
2.Add 500μl Trizol, vortex to mix thoroughly and let the tube sit at room temperature for 10 min.
3.Centrifuge at 12,000 x g for 10 min at 4℃.Transfer the supernatant into a new 1.5ml EP tube(RNase-Free).
4.Add 100μl chloroform, vigorously shake with hand for 15s and then let the tube sit at room temperature for 10 min.
5.Centrifuge at 12,000 x g for 15 min at 4℃. Transfer the upper colourless aqueous phase into a new 1.5ml EP tube(RNase-Free).
6.Add 250μl isopropanol, turn the tube upside down many times for around 10 min, and let it sit at room temperature for 10 min until the white floccule arise,and then centrifuge at 12,000 x g for 10 min at 4℃.
7.Discard the supernatant,add 500μl,75%ethanol with DEPC,and mix thoroughly.Centrifuge at 7,500 x g for 5 min at 4℃ and discard the supernatant.
8.Dry it at room temperature or in the bechtop for 15 min.
9.Add 15-20μl RNase-Free water to dissolve RNA when the precipitate become transparent.
10.Store RNA at -80℃.
RT-PCR and PCR
We amplify DNA of CSBV by RT-PCR and PCR. And the detail systems and programs are showed as bellow:
1.RT-PCR（using M-MLV Reserve Transcriptase kit bought from Promega company
ComponentVolume(μl)
Whole RNA of CSBV4.0
dNTP Mixture(10mM)4.0
Reserve primer2.0
70℃ pre-denaturation10min
Component(add in order) Volume(μl)
5×M-MuLV buffer4.0
RNase free water4.5
M-MLV Reserve Transcriptase1.0
42℃1h
72℃15mins
Store cDNA at -20℃
2.PCR
(1)System(50μl)
10×PCR Buffer5.0μl
2.5mM dNTP Mix2.0μl
Template(cDNA)2.0μl
Primer F2.0μl
Primer R2.0μl
Dream Taq DNA pol0.5μl
ddH2O36.5μl
Volume50.0μl
(2)Program
94℃5min
94℃30s
55℃(Tm-5℃)30s
72℃T(1min/kb) 2 34
72℃10min
16℃∞
qPCR
We use GoTaq? qPCR Master Mix Kit(buy from Promega company) and Eppendrof Mastercycler? ep realplex Real-Time PCR to do qPCR.
(1)System(20μl)
SYBR Premix Ex TaqTM 2×mix10.0μl
Template(cDNA)1.0μl
Primer F0.4μl
Primer R0.4μl
RNase-Free water8.2μl
Volume20.0μl
(2)Program
95℃2min
95℃20s
60℃20s
72℃30s
72℃10min
16℃∞
Plasmid Extraction
We use E.Z.N.A. Plasmid DNA Mini Kit I bought from OMEGA bio-tek company to
extract plasmids and the detail protocol is showed as bellow:
1. Isolate a single colony from a freshly streaked selective plate, and inoculate a culture of 1- 5 mL LB medium containing the appropriate selective antibiotic. Incubate for ~12-16 hours at
37°C with vigorous shaking (~ 300 rpm).
2. Centrifuge at 10,000 x g for 1 minute at room temperature
3. Decant or aspirate and discard the culture media.
4. Add 250 mL Solution I/RNase A. Vortex or pipet up and down to mix thoroughly.
5. Transfer suspension into a new 1.5 mL microcentrifuge tube.
6. Add 250 mL Solution II. Invert and gently rotate the tube several times to obtain a clear lysate. A 2-3 minute incubation may be necessary.(Avoid vigorous mixing)
7. Add 350 mL Solution III. Immediately invert several times until a flocculent white precipitate forms.
8. Centrifuge at maximum speed (≥13,000 x g) for 10 minutes. A compact white pellet will form. Promptly proceed to the next step.
9. Insert a HiBind DNA Mini Column into a 2 mL Collection Tube.
10. Transfer the cleared supernatant from Step 8 by CAREFULLY aspirating it into the HiBind DNA Mini Column. Be careful not to disturb the pellet and that no cellular debris is transferred to the HiBind DNA Mini Column.
11. Centrifuge at maximum speed for 1 minute.
12. Discard the filtrate and reuse the collection tube.
13. Add 500 mL HB Buffer.
14. Centrifuge at maximum speed for 1 minute.
15. Discard the filtrate and reuse collection tube.
16. Add 700 mL DNA Wash Buffer.
17. Centrifuge at maximum speed for 1 minute.
18. Discard the filtrate and reuse the collection tube.
19. Centrifuge the empty HiBind DNA Mini Column for 2 minutes at maximum speed to dry the column matrix.
20. Transfer the HiBind DNA Mini Column to a clean 1.5 mL microcentrifuge tube.
21. Add 30-100 μl Elution Buffer or sterile deionized water directly to the center of the column membrane.
22. Let sit at room temperature for 1 minute.
23. Centrifuge at maximum speed for 1 minute.
24. Store DNA at -20°C.
Gel Extraction
We use E.Z.N.A. Gel Extraction Kit bought from OMEGA bio-tek company to recover our target fragments after AGE and the detail protocol is showed as bellow:
1. Perform agarose gel/ethidium bromide electrophoresis to fractionate DNA fragments. Any type or grade of agarose may be used. However, it is strongly recommended that fresh TAE buffer or TBE buffer be used as running buffer. Do not reuse running buffer as its pH will increase and reduce yields.
2. When adequate separation of bands has occurred, carefully excise the DNA fragment of interest using a wide, clean, sharp scalpel. Minimize the size of the gel slice by removing extra agarose.
3. Determine the appropriate volume of the gel slice by weighing it in a clean 1.5 mL microcentrifuge tube. Assuming a density of 1 g/mL, the volume of gel is derived as follows: a gel slice of mass 0.3 g will have a volume of 0.3 mL.
4. Add 1 volume Binding Buffer (XP2).
5. Incubate at 60°C for 7 minutes or until the gel has completely melted. Vortex or shake the tube every 2-3 minutes.
6. Insert a HiBind DNA Mini Column in a 2 mL Collection Tube.
7. Add no more than 700μl DNA/agarose solution from Step 5 to the HiBind? DNA Mini Column.
8. Centrifuge at 10,000 x g for 1 minute at room temperature.
9. Discard the filtrate and reuse collection tube.
10. Repeat Steps 7-9 until all of the sample has been transferred to the column.
11. Add 300 mL Binding Buffer (XP2).
12. Centrifuge at maximum speed (≥13,000 x g) for 1 minute at room temperature.
13. Discard the filtrate and reuse collection tube.
14. Add 700μl SPW Wash Buffer.
15. Centrifuge at maximum speed for 1 minute at room temperature.
16. Discard the filtrate and reuse collection tube.
17. Centrifuge the empty HiBind DNA Mini Column for 2 minutes at maximum speed to dry the column matrix.
18. Transfer the HiBind DNA Mini Column to a clean 1.5 mL microcentrifuge tube.
19. Add 30-50μl Elution Buffer or deionized water directly to the center of the column membrane.
20. Let sit at room temperature for 2 minutes.
21. Centrifuge at maximum speed for 1 minute.
22.Store DNA at -20°C.
CTAB Extraction of dsRNA
1.Pipet 2mL bacterium suspension, centrifuge at 5,000rpm for 1 minute.
2.Discard the supernatant, add 500μl TE Buffer(10mmol· L-1Tris, 1mmol· L-1EDTA,PH7.5) and resuspend.
3.Add 30μl 10%SDS,mix thoroughly and put it in 37℃ incubator for 1h.
4.Add 100μl NaCl(5mol· L-1), mix thoroughly.
5.Add 80μl CTAB/NaCl, mix thoroughly, 65℃ water bath for 10min.
6.Add isometric phenol/ chloroform/isoamylol mixture(25:24:1), mix thoroughly and centrifuge at 12,000rpm for 5 minute at 4℃.
7.Transfer the supernatant into a new 1.5 mL microcentrifuge tube. Add 3/5 volume of it isopropanol. Centrifuge at 12,000rpm for 10 minute at 4℃.
8.Discard the supernatant, add 500μl 75% ethanol, vortex to mix thoroughly, and centrifuge at 6,000rpm for 5 minute at 4℃.
9.Vacuum dry for 10minute, and digest with DNase(1U), RNase(1U) respectively.
10.Store dsRNA at -80℃ or use for next experiment immediately.
Dual-enzyme digestion
1.Vector 10.0μl
Restriction enzyme(EcoR1,Pst1) 0.5μl
Buffer(10×H) 2.0μl
ddH2O 7.0μl
Volume 20.0μl
37℃ metal bath,8h or overnight.
2.Recover target fragments by gel extraction.
Linkage and transformation
1.Linkage system(10μl): Target fragment 4.0μl, Vector 1.0μl, 5×Buffer 2.0μl, T4 ligase 1.0μl, ddH2O 2.0μl. 4℃, overnight.
2.Add the product of linkage into 50μl DH5αcompetent cell, pipet up and down gently to mix thoroughly, and put it on ice for 30min.
3.Heat in 42℃ water bath for 90s, and take it out immediately and put it on ice for at least 2min.
4.Add 200μl LB medium without any selective antibiotic. Incubate for 45min at 37℃with vigorous shaking (~ 220 rpm).
5.Pipet 150μl bacterium suspension and smear it on LB plate with the appropriate selective antibiotic. Culture in 37℃ incubator,overnight.