实验概要
The E.Z.N.A.® Yeast RNA Kit allows convenient isolation of high-quality total RNA from a wide variety of yeast species. Up to 2 x 107 log-phase cultured yeast cells can be processed. The system combines the reversible nucleic acid-binding properties of HiBind® matrix with the speed and versatility of spin column technology to yield approximately 30 ug of RNA, with an A260/A280 ratio of 1.7-1.9. Purified RNA is suitable for downstream applications such as RT-PCR, DD-PCR, and hybridization techniques. There are no organic extractions, thus reducing plastic waste and hands-on time to allow multiple samples to be processed in parallel.
主要试剂
1. 70% ethanol - do not use other alcohols.
2. Absolute ethanol (96-100%)
3. 2-Mercaptoethanol
主要设备
1. Microcentrifuge and nuclease-free 2.0 and 1.5 ml tubes.
2. Swinging-bucket centrifuge and sterile 15 ml tubes.
3. Waterbath equilibrated to 30°C.
实验步骤
This protocol uses enzymatic lysis to prepare spheroblasts. Have the following reagents and supplies ready:
While the HiBind® RNA mini columns can bind up to 100 ug RNA, for effective purification, use no more than 2 x 107 log-phase yeast cells. For S cerevisiae grown in YPD, an OD600 of 1.0 corresponds to approximately 2 x 107 cells per ml.
1. Collect no more than 2 x 107 yeast cells in a 15 ml tube by centrifuging for 5 min at 1000 x g at 4°C.
NOTE: Use only freshly harvested cells for preparation of spheroblasts.
2. Aspirate and discard supernatant completely and resuspend cells in 2.0 ml Buffer SE /Lyticase mixture. Incubate at 30°C for 30 min, inverting the tube once every 10 min.
3. Pellet spheroblasts by centrifuging 5 min at 400 x g at room temperature. Carefully aspirate and discard supernatant. Incomplete removal of supernatant will prevent complete lysis of spheroblasts in the next step.
4. Add 350 ul of Buffer YRL/2-mercaptoethanol and 50 mg glass beads into the tube. Vortex at maxi speed for 5 minutes to lyse and homogenize the sample. If bead mill is available, lyse the cell with bead mill at top speed until the cells are completely lysed.
5. Centrifuge at 13,000 x g for 3 min. Transfer the supernatant into a new centrifuge tube.
6. Add an equal volume 70% ethanol to the lysate and mix thoroughly by pipetting. A white precipitate may form upon addition of ethanol; it will not interfere with the procedure. Do not centrifuge the tube.
7. Apply the entire sample (around 700 ul) to a HiBind® RNA Mini column assembled in a 2 ml collection tube (supplied). Centrifuge at $10,000 x g for 30-60 seconds at room temperature. Discard flow-through and re-use collection tube in Step 8 or Step 9.
Note: This is the point at which optional on-membrane DNase I digestion should begin. If DNase I digestion is desired, skip Step 8 and proceed to Step 9. If DNase I digestion is not needed, proceed with Step 8.
8. Wash the sample by adding 700ul of RNA Wash Buffer I to the column. Centrifuge at 10,000 x g for 30-60 seconds at room temperature. Discard the flow-through and re-use the collection tube.
9. DNase I Digestion (Optional)
Since HiBind® RNA resin and spin-column technology actually removes most DNA without the DNase treatment, this DNase I digestion step is not necessary for most downstream applications. However, certain sensitive RNA applications might require further DNA removal. (See DNase I, Cat # E1091 for detailed information.)
1) Wash the column by adding 300ul of RNA Wash Buffer I to the column. Centrifuge at 10,000 x g for 30-60 seconds at room temperature. Discard the flow-through and re-use the collection tube.
2) For each HiBind® RNA column, have the following DNase I digestion reaction mixture prepared in advance as follows:
OBI DNase I Digestion Buffer 73.5 ul
RNase-free DNase I (20 Kunitz unites/ul) 1.5 ul
Total volume 75 ul
Note:
a. DNase I is very sensitive, and is thus subject to physical denaturation, so do not vortex this DNase I mixture. Mix gently by inverting the tube. Prepare the fresh DNase I digestion mixture before RNA isolation.
b. OBI DNase I digestion buffer is supplied with the OBI RNase-free DNase set.
c. Standard DNase buffers may not be compatible with the Omega Bio-Tek Kit for on-membrane DNase digestion.
3) Dry column by spinning an additional 30 seconds, then pipet 75 ul of the DNase I digestion reaction mixture directly onto the surface of the HiBind® RNA membrane in each column. Make sure to pipet the DNase I digestion mixture directly onto the membrane. DNase I digestion might not be complete if some of the mixture sticks to the wall or to the O-ring of the HiBind® RNA column.
4) Incubate at room temperature(25-30°C) for 15 minutes.
5) Place column in a clean 2 ml collection tube and add 400 ul RNA Wash Buffer I. Allow wash buffer to soak column for at least 5 minutes before proceeding. Centrifuge at 10,000 x g for 15 seconds at room temperature. Discard flow-through and re-use the collection tube in next step.
10. Place column in the same 2 ml collection tube, and add 500 ul RNA Wash Buffer II diluted with ethanol. Centrifuge at 10,000 x g for 30-60 seconds and discard flow-through. Re-use the collection tube in next step.
Note: RNA Wash Buffer II Concentrate must be diluted with absolute ethanol before use. Refer to Page 4 or to label on bottle for directions.
11. Wash sample with a second 500 ul of RNA Wash Buffer II and centrifuge and discard flow-through as in preceding step. Using the same collection tube, centrifuge the spin cartridge at 10,000 x g for 2 min at full speed to completely dry the HiBind® matrix.
12. Elution of RNA. Transfer the column to a clean 1.5 ml microfuge tube (not supplied with kit) and elute the RNA with 30-50 ul of DEPC-treated water (supplied with kit). Make sure to add water directly onto column matrix. Centrifuge 1 min at maximum speed. A second elution may be necessary if the expected yield of RNA is greater than 50 ug. Alternatively, RNA may be eluted with a greater volume of water. While additional elutions increase total RNA yield, the concentration will be lowered since more than 80% of RNA is recovered with the first elution. Pre-heating the water to 70°C before adding to column and incubating column 5 min at room temperature before centrifugation may increase yields.