BISC311:Double-stranded RNA synthesis
Step 7 Send off to have the insert sequenced Check sequence to make sure that NOT1 and Spe1 restriction enzymes don’t cut the sequences.
Step 8 Restriction reaction
For restriction digest: Digest Spe1 Not1 Plasmid 60 60 Buffer (#2 for Spe I and #3 for Not I) 14 14 Enzyme 5 5 Water 61 61 Total 140 140
Incubate at 37C O/N (~16hrs)
• Run 2ul of product on gel to check that the plasmid has been cut. Make sure there is only 1 band.
• Add same amount (140 µl) of phenol/chloroform (for DNA i.e. Tris saturated pH 8.0) and vigorously vortex. Keep for 5-15 min on ice.
• Centrifuge (14000 rpm, 10 min)
• Transfer supernatant to new tube, and add same amount of (140 µl) chloroform. Vigorously vortex.
• Centrifuge (14000 rpm, 10 min)
• Transfer supernatant to new tube, and add 10 % volume (14 µl) of 3 M sodium acetate (pH 5.2). and either same amount (140 µl) of Isopropanol or 2-2.5 volume (280-350 µl) of ethanol.
• Keep at –20ºC at least 1 h.
• Centrifuge (14000 rpm, 10 min)
• Remove supernatant, and rinse pellet with 70-80 % ethanol.
• Centrifuge (14000 rpm, 10 min)
• Remove supernatant and air dry pellet (10-15 min).
• Dissolve into 10 µl DEPC water.
• SPEC
• Calculate the amount of plasmid DNA you will need for RNA synthesis.
➢ We need 1ug
Step9: RNA synthesis: Megascript reaction Get the tubes out on ice (only covered tubes). KEEP ENZYME IN FREEZER UNTIL NEEDED! Thaw tubes and vortex and spin down briefly. DO NOT vortex enzyme. KEEP 10X REATION BUFFER OUT and put rest of tubes back on ice.
RNA synthesis • MEGAscript T3 and T7 Restriction enzymes FOR T3: NOT1 FOR T7: SPE1
Use RNAase free tube
Set up reaction • Reaction example
| Reagent | Amount in T3 | Amount in T7 |
|---|---|---|
|
1ug Plasmid digested by Not I |
X µl | |
| 1ug Plasmid digested by Spe I |
|
XuL |
| ATP solution |
2 µl |
2 µl |
| CTP solution |
2 µl |
2 µl |
| GTP solution |
2 µl |
2 µl |
| UTP solution |
2 µl |
2 µ |
| 10xBuffer |
2 µl |
2 µl |
| T3 Enzyme Mix |
2 µl | |
| T7 Enzyme Mix |
|
2 µl |
| MilliQ |
8-X µl |
8-X µl |
| TOTAL |
20 µl |
20 µl| -|}
• Incubate at 37ºC overnight. Step 10 Clean the RNA • Keep 1-2 µl of RNA solution for gel (keep at –20ºC). • Add 230 µl of DEPC water and 30 µl of Ammonium acetate, which is in MEGAscript kit. • Add same amount (250 µl) of phenol/chloroform (for RNA i.e. water saturated approx. pH 4.0) and vigorously vortex. Vortex for 2 minutes, with caps tightly on and using 2 tubes at a time to increase vigor. • Keep for 5-15 min on ice. • Centrifuge (14000 rpm, 10 min) • Get RNAse free tubes for the RNAi • Pull out the tubes from the centrifuge carefully – need the top layer • Transfer supernatant to new tube, and add same amount of (250 µl) chloroform. o Chloroform in hood (pipette up and down to minimize dripping). • Vigorously vortex. Vortex for 2 minutes, with caps tightly on and using 2 tubes at a time to increase vigor. • Centrifuge (14000 rpm, 10 min) • Transfer supernatant to new tube, and add same amount (250 µl) of Isopropanol. Don’t pull up all top layer at once. • Keep at –20ºC at least 1 h (overnight increases yield). • Centrifuge (14000 rpm, 10 min) • Remove supernatant, and rinse pellet with 70-80 % ethanol in DEPC water. • Centrifuge (14000 rpm, 10 min) • Remove supernatant and air dry pellet (10-15 min). Dry till most of the liquid is gone and the pellet sort of looks clear. Do not overdry! • For large RNA pellet, add 30ul (the more concentrated the better for RNAi as you don’t need to inject as much) of DEPC water. • SPEC
You need to calculate these values. Below is an example of the calculation you need to perform: Example Calculation of total amount of RNA you have: 2818ng/ul X 30uL=84540ng Spe 1: 84540 ng Not 1: 168420 ug Limiting reagent: spe1 Calculation of the amount of RNA you should add: 84540ug/5614ug/ul=15.05 uL of NOT 1 Calculation of the amount of DEPC water you nee to add to the mixture to make 2 µg/ µl solution Spe1 30 µl Not 1 15.05 µl 169.08 µg double strand RNA in 45.05 uL ---> Add 39.49 µl ---> annealing reaction ---> keep at -80 ºC Step 11 Annealing reaction On the PCR machine: Option->Create->Method->147-148
Run gel |
