Oligo (dT) magnetic beads have a suitable Oligo (dT) loading capacity, with a size of approximately 1 μ m. Good dispersibility, high magnetic content, and fast magnetic response speed. By complementary pairing between Oligo dT on the surface of magnetic beads and Ploy A at the tail of mRNA, complete and high-purity mRNA can be efficiently separated from total RNA in eukaryotes or directly from plant tissues and cells. The extracted mRNA can be used in various downstream experiments in molecular biology, such as RT-PCR, cDNA library construction, Northern Blot analysis, primer extension, gene expression analysis, etc.
[Operation Steps]
1. Prepare buffer solution and materials
1.1 Preparation of buffer solution: Users can adjust the buffer solution formula according to their needs (all reagents need to be prepared with DEPC treated water)
Binding buffer
100 mM Tris HCl, pH7.5
500 mM LiCl
10 mM EDTA, pH8
1% LiDS
5 mM dithiothreitol
Washing buffer A
10 mM Tris HCl, pH 7.5
0.15 M LiCl
1 mM EDTA
0.1% LiDS
Washing buffer B
10 mM Tris HCl, pH 7.5
0.15 M LiCl
1 mM EDTA
Eluent
10 mM Tris HCl pH 7.5
Note: All reagents should be balanced to room temperature during use. If there is precipitation, it can be preheated at 37 ℃ for 10 minutes.
1.2 RNase free 1.5 mL centrifuge tube;
1.3 Magnetic holder for 1.5 mL centrifuge tube;
1.4 Diethyl pyrocarbonate (DEPC) water: Add DEPC with a final concentration of 0.1% (V/V) to purified water, mix well, and leave overnight for high-pressure sterilization.
1.5 Water bath pot;
1.6 Vortex oscillator
1.7 Rotary Mixer or Shaker
2. Clean Oligo (dT) magnetic beads
2.1 Thoroughly resuspend the Oligo (dT) magnetic beads in the reagent bottle (shake the shaker for 10 minutes or manually shake until fully mixed).
2.2 Transfer the required volume of Oligo (dT) magnetic beads to a centrifuge tube and add binding buffer to achieve a magnetic bead concentration of 0.4 mg/mL.
2.3 Place the centrifuge tube on a magnetic rack for 1 minute and discard the upper cleaning solution.
When removing the upper cleaning agent, try to remove the residual liquid from the pipe cover and bottom as much as possible, and do not inhale magnetic beads.
2.4 Remove the centrifuge tube from the magnetic holder, add a binding buffer of the same volume as the initial volume, and keep it as a backup for the next step.
3. Purification of mRNA from Total RNA
3.1 After thoroughly mixing the magnetic beads washed and resuspended with a combined buffer, take 50 μ L into the centrifuge tube.
3.2 Sample preparation: Use DEPC water to mix 10 μ Adjust the volume of the total RNA sample to 50 μ L. Add 50 μ In the centrifuge tube of the L magnetic bead, gently mix with a pipette.
3.3 Place the sample in the PCR apparatus at 65 ℃ for 5 minutes, 25 ℃ for 5 minutes, and 4 ℃ for hold.
3.4 Magnetic separation, remove supernatant, use 200 μ Wash the magnetic beads bound to mRNA with L washing buffer A, gently mix with a pipette, magnetic separation, and remove the supernatant.
Attention points:
When completing the cleaning, be sure to remove all washing buffer.
3.5 Add 50 μ L eluent, gently mix with a pipette, place the sample in a PCR apparatus at 80 ℃ for 2 minutes, and hold at 25 ℃.
3.6 Add 50 μ L combined solution, gently mix with a pipette, and let stand at room temperature for 5 minutes; Re magnetic separation, remove the supernatant.
3.7 Add 200 μ L washing solution buffer A, gently mix with a pipette, magnetic separation, and remove the supernatant.
Attention points:
When completing the cleaning, be sure to remove all washing buffer.
3.8 Add 200 μ L washing solution buffer B, gently mix with a pipette, magnetic separation, and remove the supernatant. Repeat this step twice.
Attention points:
When completing the cleaning, be sure to remove all washing buffer.
3.9 Remove the centrifuge tube from the magnetic support and add 10 μ L DEPC water or eluent, resuspended magnetic beads, 80 ℃ for 2 minutes.
3.10 Quickly use a magnetic rack to collect the supernatant and transfer it to a centrifuge tube without RNase for downstream experiments.
【 Precautions 】
1. Minimize RNA degradation as much as possible; When using RNA, please always wear gloves to minimize RNase contamination; All buffers and consumables used for mRNA extraction should be RNase free.
To reduce the loss of magnetic beads, each magnetic separation time should not be less than 1 minute.
3. Thoroughly resuspend the magnetic bead/mRNA complex during the washing process, and completely remove the washing buffer at each step to prevent LiDS and other salts from being carried downstream in the reaction. LiDS is a strong inhibitor of enzymatic reactions.
If the residual amount of purified rRNA is too high for downstream applications, it is recommended to use new magnetic beads for the second round of mRNA purification.
5. This product is for research use only.
