Transportation and preservation
Normal temperature
transport
And storage, but RNase-free DNase and DNase membrane reaction solution need to be stored at -20 °C, valid for one year
Self-prepared reagent
Instructions
1.
Estimate the amount of tissue cells used. Each micro-extraction typically requires 100-200 mg of plant leaves
,or
50-100 mg plant seeds
,or
200-500 mg plant fruit.
2.
Sample broken:
1)
Homogenization method:
Fresh first
plant
Tissue cut into small pieces (stored in RNA
LOCKER
middle
plant
Tissue needs to use paper to remove RNA
LOCKER
After liquid
again
Cut into small pieces)
,
put into a
10-15 mL plastic centrifuge tube,
Add 1 mL of solution A
, of course
Homogenizer
5-20 seconds.
Foaming occurs when homogenizing
,
But it does not affect the extraction effect.
2)
Liquid nitrogen grinding method (suitable for complex, easily degradable samples): Take appropriate amount of fresh plant tissue into a liquid nitrogen-containing mortar, quickly grind the tissue into a powder, and transfer the powder to a suitable plastic centrifuge tube.
Add 1 mL of solution A
, immediately shake vigorously for 20 seconds, mix thoroughly.
note:
The solution A was precipitated.
If put
Solution A
Put
Placed at 4 ° C,
a period of time
Rear
may
Will produce precipitation
In this case
It must be placed in a 65 ° C water bath to completely dissolve the precipitate before use.
Shake well and then take it again
.
3.
Homogenate
Object or research
mill
Object
Transfer to
Clean 1.5 mL plastic
In a centrifuge tube (no need to transfer non-liquid cell debris). Some plant tissues (
such as
Fruit) contains a lot of water
,
Homogenate
meeting
More than 1 m
L,
Only 1 m is taken when transferring
L
.
4.
Add 0.3 mL of Solution B and 0.2 mL to your tube.
,in
Oscillator
on
The mixture was shaken for 30 seconds, and the solution was uniformly emulsified. The bottom of the tube must be shaken when the oscillator oscillates.
5.
Room temperature
12000 rpm
Centrifugation
3-
5 minutes
,
There will be an agreement between the two phases
5
Millimeter thick cell disrupted material.
6.
The supernatant will be (about 0.
6
mL) transfer to another clean
1.5 mL plastic
Centrifuge tube
,
Lower organic phase and intermediate layer contain DNA
,
Protein and other impurities
,
Avoid touching or sucking. It is best to leave 100 uL of supernatant without taking it.
7.
Add an equal volume of solution C to the supernatant and mix thoroughly
.
If a precipitate is produced (for some plants, it is normal), do not remove the precipitate, be sure to put all the mixture on the column.
8.
Transfer half of the mixture (containing sediment, if any) to the centrifugal column,
12000 rpm
Centrifuge at room temperature
half
minute
Discard the penetrating fluid
.
9.
Transfer the remaining half of the mixture (containing sediment, if any) to the same centrifugal column.
12000 rpm
Centrifuge at room temperature
half
minute
Discard the penetrating fluid
.
10.
Add 0.7 mL of universal wash column,
12000 rpm
Centrifuge at room temperature
half
minute
Discard the penetrating fluid
.
again
plus
0.3 mL universal wash column,
12000 rpm
Centrifuge at room temperature
half
minute
Discard the penetrating fluid
.
Generally, the sample can be washed twice, but for the sample which is precipitated after adding the solution C in the seventh step, it needs to be washed three times, and the amount of the common washing liquid added each time is 0.4, 0.3 and 0.3 mL, respectively. constant.
11.
Centrifuge at 12000 rpm for 10 seconds at room temperature to remove residual liquid. This step is important, otherwise the residual universal wash column will inhibit the activity of DNase in subsequent reactions.
12.
10 uL (10 U) of RNase-free DNase was added to a 50 uL 37 ° C preheated DNA membrane reaction solution, and mixed with a DNase working solution by pipetting and mixing.
13.
The DNase working solution was preheated at 37 ° C for 1 minute, then all was added to a centrifugal adsorption column and allowed to stand at room temperature for 5 minutes. Note: 5 minutes is generally sufficient to degrade DNA contamination in most cases. If the DNA is not completely degraded (possibly because the residual impurities in the sample inhibit the activity of DNase), the incubation time of this step can be extended to 10 minutes or 15 minutes.
14.
Directly in the centrifugal adsorption column
Add 0.7 mL of universal wash column, cover and cover and mix for several times.
15.
12000 rpm
Centrifuge at room temperature
half
minute
Discard the penetrating fluid
.
16.
Plus
0.3 mL universal washing column into the centrifugal adsorption column,
12000 rpm
Centrifuge at room temperature
half
minute
Discard the penetrating fluid
.
17.
12000 rpm
Centrifuge at room temperature
half
minute. This step is very important, otherwise residual ethanol
(ingredients in general washing column)
Will affect the use of RNA.
18.
Transfer the centrifugal adsorption column to the RNase-free collection tube and add 30-50 uL RNA eluate to the chamber.
temperature
Place 1-2
minute
.
19.
12000 rpm
Centrifuge at room temperature
half
minute
The solution in the centrifuge tube is RNA
sample.
The centrifugal adsorption column provided by this product has strong adsorption force. It is not possible to wash all the RNA on the membrane by one elution. If necessary, add 30-50 uL RNA eluent once.
20.
The resulting RNA solution can
Use immediately or store at -80 ° C for use.
If you want to proceed
Formaldehyde denaturing electrophoresis
Detection. If non-denaturing gel electrophoresis is used, RNAon loading solution must be used. Never use a DNA sample (because it is generally not treated with RNase).
related products
mRNA extraction kit (CAT#:80817)
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