Principle:
The QIAamp Viral RNA Mini Kit combines the selective binding properties of a silica-gel-based membrane with the speed of a microspin. The sample is first lysed under high denaturing conditions to inactivate RNases and to ensure isolation of intact viral RNA. Buffering conditions provide optimum binding of the RNA to the QIAamp membrane, and the sample is loaded onto the QIAamp mini spin column. The RNA binds to the membrane, and contaminants are efficiently washed away using two different was buffers. High quality RNA is eluted in a special RNAse-free buffer ready for direct use or safe storage.
The QIAamp Viral RNA Mini Kit combines the selective binding properties of a silica-gel-based membrane with the speed of a microspin. The sample is first lysed under high denaturing conditions to inactivate RNases and to ensure isolation of intact viral RNA. Buffering conditions provide optimum binding of the RNA to the QIAamp membrane, and the sample is loaded onto the QIAamp mini spin column. The RNA binds to the membrane, and contaminants are efficiently washed away using two different was buffers. High quality RNA is eluted in a special RNAse-free buffer ready for direct use or safe storage.
Precautions:
- Buffers AVL and AW1 contain guanidine salts, which can form highly reactive compounds when combined with bleach. For spillage, clean with detergent and water.
- Avoid touching the QIAmp membrane with the pipet tip.
- Wear powder-free gloves throughout the entire procedure.
- Prepare Buffer AVL- Carrier RNA mixture.See table below:
No. Of Samples
|
Vol. Buffer AVL(ml)
|
Vol. Carrier RNA-AVE (ul)
|
No. Of Samples
|
Vol. Buffer AVL(ml)
|
Vol. Carrier RNA-AVE (ul)
|
1
|
0.56
|
5.6
|
13
|
7.28
|
72.8
|
2
|
1.12
|
11.2
|
14
|
7.84
|
78.4
|
3
|
1.68
|
16.8
|
15
|
8.40
|
84.0
|
4
|
2.24
|
22.4
|
16
|
8.96
|
89.6
|
5
|
2.80
|
28.0
|
17
|
9.52
|
95.2
|
6
|
3.36
|
33.6
|
18
|
10.08
|
100.8
|
7
|
3.92
|
39.2
|
19
|
10.64
|
106.4
|
8
|
4.48
|
44.8
|
20
|
11.20
|
112.0
|
9
|
5.04
|
50.4
|
21
|
11.76
|
117.6
|
10
|
5.60
|
56.0
|
22
|
12.32
|
123.2
|
11
|
6.16
|
61.6
|
23
|
12.88
|
128.8
|
12
|
6.72
|
67.2
|
24
|
13.44
|
134.4
|
- Pipet 560 ul of prepared Buffer AVL containing carrier RNA into a 1.5 ml microcentrifuge tube.
- Add 140 ul of sample and mix by pulse-vortexing for 15 seconds.
- Incubate at room temperature (15-250C) for 10 minutes.
- Briefly centrifuge the tube to remove drops from the inside of the lid.
- Add 560 ul of ethanol to the sample and mix by pulse-vortexing for 15 seconds.
- Briefly centrifuge the tube to remove drops from the inside lid.
- Carefully apply 630 ul of the solution from step 5 to the QIAamp mini spin column. Close the cap and centrifuge at 8,000 rpm for 1 minute. Place the spin column into a clean 2 ml collection tube, and discard the tube containing the filtrate.
- Carefully open the spin column, and repeat step 6.
- Carefully open the spin column, and add 500 ul of Buffer AW1. Close the cap and centrifuge at 8,000 rpm for 1 min. place the column in a clean 2 ml collection tube and discard the tube containing the filtrate.
- Add 500 ul Buffer AW2 and centrifuge at 14,000 rpm for 3 min.
- (Optional)Place spin column in a new 2 ml collection tube and centrifuge full speed for 1 min. (extra spin).
- Place the spin column in a new 1.5 ml micro centrifuge tube. Discard the old collection tube containing the filtrate. Carefully open the spin column and add 60 ul of Buffer AVE equilibrated to room temperature. Close the cap and incubate at room temperature for 5 min, centrifuge at 8,000 rpm for 1 min.
- Eluate now ready for PCR.
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