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RNA Miniprep Protocol - Yeast

1.  Grow cells in 3 ml selective media overnight at an appropriate temperature to an OD600 > 1.

2.       Pellet the cells in 1.5 ml microtubes for 2 min at 14,000 gin a table-top microcentrifuge.
 
3.       Resuspend the pellet in 100 µl of the following solution:
1 M Sorbitol
            0.1 M EDTA (pH 7.4)
      Just before use, add 0.1% β-mercaptoethanol (final concentration) and 50 units Zymolase.
 
4.       Incubate at 30°C for 15-30 min until the solution appears clear.
 
5.       Add 400 µlBuffer LY. Mix gently.
 
6.       Transfer the solution into the binding column and centrifuge at 14,000 rpm for 1 min. Discard the collection tube with the flow-through and put the column back to a new collection tube.
 
Without DNase I treatment:
 
7.       Add400 µL Buffer RBto the column and centrifuge at 13,000 rpm for 30s. Discard the collection tube with the flow-through. Put the column back to a new collection tube.
 
8.       Add 500 µL RNA Wash Bufferto the column and centrifuge at 13,000 rpm for 30 s. Discard the flow-through. Repeat once. Ensure that ethanol has been added to RNA wash buffer before use.
 
9.       Centrifuge the column with the lid open at 13,000 rpm for 1 min. It is critical to remove residue ethanol for optimal elution.
 
10.   Transfer the column to an RNase-free 1.5 mL tube. Add 50 µL DEPC-treated ddH2O to the center of the column. Centrifuge at 13,000 rpm for 1 min to elute the RNA. Store the RNA solution at -20 °C.
 
With DNase I treatment:
 
7.       Add 500 µL RNA Wash Bufferto the column and centrifuge at 13,000 rpm for 30 s. Discard the flow-through. Ensure that ethanol has been added to RNA wash buffer before use.
 
8.       Add 75 µL DNase I (5URNase-free) with Digestion Buffer onto the middle of the column and incubate at room temperature for 15 min. Add 400 µL Buffer RB to the column, incubate for 1-2 min, and centrifuge at 13,000 rpm for 30s. Discard the collection tube with the flow-through. Put the column back to a new collection tube.
 
9.       Add 500 µL RNA Wash Bufferto the column and centrifuge at 13,000 rpm for 30 s. Discard the flow-through. Repeat once. Ensure that ethanol has been added to RNA wash buffer before use.
 
10.  Centrifuge the column with the lid open at 13,000 rpm for 1 min. It is critical to remove residue ethanol for optimal elution.

11.  Transfer the column to an RNase-free 1.5 mL tube. Add 50 µL DEPC-treated ddH2O to the center of the column. Centrifuge at 13,000 rpm for 1 min to elute the RNA. Store the RNA solution at -20 °C.

 
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