Ver. L0918
L0918
Total RNA Extraction Mini Kit (Blood/Cultured Cells)
(SYG301-001 (Sample); FYG301-50P (50 preps); FYG301-100P (100 preps); FYG301-300P (300 preps))
Store at room temperature (15ºC-25 ºC)
Sample:
up to 300 μl of whole blood
Yield: 5-30 μg
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Format: spin column
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Operation time: within 30 minutes
up to 5 x 10 cultured animal cells
up to 1 x 10 cultured bacterial cells
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up to 5 x 10 fungus cells
Description
The kit is specially designed for purification of total RNA from cultured cells and fresh human
whole blood. The method use detergents and a chaotropic salt to lyse the cells and inactivate
RNase, than RNA in chaotropic salt solutions binds to the glass fiber matrix of the columns. After
washing off the contaminants, the purified RNA is eluted by RNase-free water. The entire
procedure can be completed within 30 minutes and the purified RNA is ready for RT-PCR,
northern blotting, primer extension and cDNA library construction.
Component
SYG301-001
FYG301-50P
FYG301-100P
FYG301-300P
(Sample)
(50 preps)
(100 preps)
(300 preps)
10 ml
100 ml
200 ml
500 ml
RB Buffer
2 ml
30 ml
60 ml
130 ml
RT Buffer
1.5 ml
15 ml
30 ml
75 ml
WI Buffer
2 ml
30 ml
50 ml
130 ml
Wash Buffer*
1 ml
12.5 ml
25 ml
50 ml +50 ml
(Add ethanol)
(4 ml)
(50 ml)
(100 ml)
(100 ml + 100 ml)
RNase-Free Water
1 ml
6 ml
6 ml
30 ml
RB Column
4 pcs
50 pcs
100 pcs
300 pcs
2 ml Collection Tube
8 pcs
100 pcs
200 pcs
600 pcs
RBC Lysis Buffer
*Add 4 ml (SYG301-001) / 50 ml (FYG301-50P) / 100 ml (FYG301-100P) / 200 ml (FYG301-300P)
ethanol (96~100%) to Wash Buffer before first use.
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Blood/ Cultured Cell Protocol
․Add ethanol (96~100%) to Wash Buffer prior to initial use.
․Additional requirements: PBS
70% Ethanol
1.5 ml microcentrifuge tube
pipette tips (RNase-free)
ß-mercaptoethanol
0.10-0.25% Trypsin
DNase I: DNase I (RNase free) 2 KU/ml in reaction buffer (50μg/ml BSA, 50 mM Tris-HCl,
10 mM MnCl2, pH 7.0 at 25ºC).
Step 1. RBC Lysis/Cell Harvesting
a. Use fresh human blood:
i. Collect fresh human blood in anticoagulant-treated collection tubes.
ii. Add 1 ml RBC Lysis Buffer to a sterile 1.5 ml reaction tube. (RNase-free)
iii. Add 300 µ l human whole blood and mix by inversion.
iv. Incubate the tube for 10 min on ice, vortex 1~2 sec. twice during incubation.
v. Centrifuge at 4℃ for 5 min at 3000 × g in a microcentrifuge.
vi. Discard the supernatant and resuspend the cells in 100 l RBC Lysis Buffer by
flicking the tube.
b. Use Cultured animal cells:
i. If using adherent cells, trypsinize the cells before lysis.
ii. Transfer up to 5 x 106 of cells to a microcentrifuge tube (not provided) and harvest
the cells with centrifugation for 5 minutes at 300 x g.
iii. Discard the supernatant and resuspend the cells in 100 l PBS or RBC Lysis Buffer.
iv. Transfer the mixture to a new microcentrifuge tube (RNase-free).
Step 2. Cell Lysis
a. Add 400 l of RB Buffer and 4 l of ß-mercaptoethanol to the white pellet from Step 1,
mix by vortexing.
b. Incubate at room temperature for 5 minutes.
Step 3. RNA Binding
a. Place a RB column in a 2 ml Collection Tube.
b. Add 500 l of 70% ethanol to the sample lysate from Step 2 and mix immediately by
pipetting (or vortexing).
c. Apply 500 l of ethanol-added mixture from previous step to the RB column.
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d. Centrifuge at full speed (14-16,000 x g) for 1 minute.
e. Discard the flow-through and apply the rest to the same RB Column.
f. Centrifuge at full speed (14-16,000 x g) for 1 minute.
g. Discard the Collection tube containing the flow-through and transfer the RB column in
a new 2 ml Collection Tube.
Optional Step: DNA residue degradation
a. Add 100 μl DNase I (2 KU/ml, not provided) to the center of the RB column matrix.
b. Stand for 10 minutes at room temperature.
c. Go to Step 4 Wash.
Step 4. Wash
a. Apply 400 l of W1 Buffer into the RB Column.
b. Centrifuge at full speed (14-16,000 x g) for 30 seconds.
c. Discard the flow-through and place the RB Column back in the 2 ml Collection Tube.
d. Add 600 l of Wash Buffer (ethanol added) into the RB Column.
e. Centrifuge at full speed (14-16,000 x g) for 30 seconds.
f. Discard the flow-through and place the RB Column back in the 2 ml Collection Tube.
g. Centrifuge at full speed (14-16,000 x g) for 3 minutes to dry the column matrix.
Step 5. RNA Elution
a.
b.
c.
d.
Place dried RB Column in a clean microcentrifuge tube (RNase-free, not provided).
Add 50 l of RNase-Free Water into the center of the column matrix.
Stand for at least 1 minute until RNase-Free Water has been absorbed by the matrix.
Centrifuge at full speed for 1 minute to elute purified RNA.
Optional Step: DNA residue degradation
a. Add 2 μl DNase I (2 KU/ml, not provided) to the final elution sample.
b. Stand for 10 minutes at room temperature.
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Bacteria Protocol
Step 1. RBC Lysis/Cell Harvesting
a. Gram-Negative Bacteria
i. Transfer the bacterial culture (up to 1 x 109) to a 1.5 ml microcentrifuge tube
(RNase-free).
ii. Centrifuge for 1 minute at 14-16,000 x g and remove the supernatant completely.
iii. Vortex the cell pellet for 30 seconds.
iv. Add 200 µl of RT Buffer to the tube and re-suspend the cell pellet by vortex or
pipetting.
v. Incubate at room temperature for 5 minutes.
b. Gram-Positive Bacteria
i. Transfer the bacterial culture (up to 1 x 109) to a 1.5 ml microcentrifuge tube
(RNase-free).
ii. Centrifuge for 1 minute at 14-16,000 x g and remove the supernatant completely.
iii. Add 200 µ l of lysozyme buffer to the tube and re-suspend the cell pellet by vortex
or pipetting.
iv. Incubate at room temperature for 10 minutes. During incubation, invert the tube
every 2-3 minutes.
Step 2. Cell Lysis
a. Add 300 l of RB Buffer and 3 µ l ß-mercaptoethanol to the sample lysate from Step 1,
mix by vortexing.
b. Incubate at room temperature for 5 minutes.
c. Centrifuge at 14-16,000 x g for 2 minutes.
d. Transfer the supernatant to a new 1.5 ml microcentrifuge tube (RNase-free).
Step 3. RNA Binding
a. Place a RB Column in a 2 ml Collection Tube.
b. Add 500 l of 70% ethanol to the sample lysate from Step 2 and mix immediately by
pipetting (or vortexing) .
c. Apply 500 l of ethanol-added mixture from previous step to the RB Column.
d. Centrifuge at full speed (14-16,000 x g) for 1 minute.
e. Discard the flow-through and apply the rest to the same RB Column.
f. Centrifuge at full speed (14-16,000 x g) for 1 minute.
g. Discard the Collection tube containing the flow-through and transfer the RB Column
in a new 2 ml Collection Tube.
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Optional Step: DNA residue degradation
a. Add 100 μl DNase I (2 KU/ml, not provided) to the center of the RB column matrix.
b. Stand for 10 minutes at room temperature.
c. Go to Step 4 Wash.
Step 4. Wash
a. Apply 400 l of W1 Buffer into the RB Column.
b. Centrifuge at full speed (14-16,000 x g) for 1 minute.
c. Discard the flow-through and place the RB Column back in the 2 ml Collection Tube.
d. Add 600 l of Wash Buffer (ethanol added) into the RB Column.
e. Centrifuge at full speed (14-16,000 x g) for 1 minute.
f. Discard the flow-through and place the RB Column back in the 2 ml Collection Tube.
g. Centrifuge at full speed (14-16,000 x g) for 3 minutes to dry the column matrix.
Step 5. RNA Elution
a. Place dried RB Column in a clean microcentrifuge tube (RNase-free, not provided).
b. Add 50 l of RNase-Free Water into the center of the column matrix.
c. Stand for 3 minutes until RNase-Free Water has been absorbed by the matrix.
d. Centrifuge at full speed for 1 minute to elute purified RNA.
Optional Step：DNA residue degradation
a.
a.
Add 2 μl DNase I (2 KU/ml, not provided) to the final elution sample.
b. Stand for 10 minutes at room temperature.
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Fungus Protocol
Step 1. Cell Harvesting
a. Centrifuge for 10 min at 5,000 × g to harvest fungus cells (up to 5 x107).
b. Discard the supernatant and resuspend the pellet in 600 l of sorbitol buffer.
c. Add 200 U of lyticase or zymolase, incubate for 30 minutes at 30℃.
d. Centrifuge the mixture for 10 minutes at 2,000 × g to harvest the spheroplast.
e. Remove the supernatant and proceed with the Lysis Step of the Bacteria Protocol.
Quality Control
The quality of Total RNA Extraction Mini Kit (Blood and Cultured cell) is tested on a lot-to-lot
basis. The Kits are tested by isolation of total RNA from fresh human whole blood and cultured
cell samples. More than 1μg of total RNA was quantified with a spectrophotometer and checked
by formaldehyde agarose gel analysis. Finally, RT-PCR was used to ensure the quality of total
RNA.
Reference
(1) Vogelstein, B., and Gillespie, D. (1979) Proc. Natl. Acad. Sci. USA 76, 615.
Note
*For research use only. Not for use in diagnostic or therapeutic procedures.
*RB Buffer contains chaotropic salt which is harmful and irritant agent. During operation, always
wear a lab coat, disposable gloves, protective goggles and procedure mask.
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