A. RNA isolation from Streptomyces using RNA protect (G.Bucca)
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Calculate the required volume of bacterial culture require for RNA isolation (we usually
extract RNA from 7.5 to 10 ml of exponentially growing cultures)
Pipette two volumes of RNA protect into a falcon tube.
Add 1 volume of bacterial culture to the tube. Vortex immediately to ensure instantaneous
quenching of the culture. Incubate for 10 minutes at room temperature (15-25°C).
Centrifuge for 10 minutes at 3500rpm.
Decant the supernatant. Remove any excess by inverting the tube onto a paper towel and
inverting once.
Samples may be frozen at –20°C for 2 weeks or –80°C for 4 weeks.
B. RNA isolation following Qiagen miniprep protocol
Materials
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Lysozyme in TE (15mg/ml)
TE buffer (10mM Tris: 1mM EDTA pH8.0)
RNaseZap
RNeasy Protect Bacteria Mini Kit PLUS
RNAprotect Bacteria Reagent
RNase-Free DNase Set
Phenol: Chloroform: Isoamyl alcohol 25:24:1 (Phenol buffered at pH8.0 with Tris)
Chloroform
96% ethanol
β-mercaptoethanol
RNase free pipette tips
RNase free eppendorfs
Pre-Preparation
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Add 10 μl β-mercaptoethanol per 1ml RLT buffer prior to use.
For the first usage of RPE add four volumes of 96-100% ethanol to the concentrate to
obtain a working solution.
Method
1.
Loosen the pellet by flicking the bottom of the tube. Resuspend the bacteria thoroughly in
200μl of lysozyme containing TE buffer by vortexing. Incubate at room temperature (or
37°C) for 10-15 minutes. Ensure there is complete digestion of the cell wall.
2.
Add 600μl buffer RLT to the sample. Vortex vigorously. Add to a 2 ml eppendorf
containing a single sterile RNase free stainless steel bead. Place in the Tissuelyser for
3.
2 mins, rotate 180o and continue for another 2 minutes. Centrifuge for 2mins and only use
the supernatant.
Add 1 volume phenol/chloroform, vortex for 30s and centrifuge for 5 minutes at full speed
@4ºC.
4.
Transfer the upper phase (aqueous) to a fresh 1.5ml eppendorf and repeat the extraction
with 1 volume phenol/chloroform. Vortex and centrifuge as in step 3.
5.
Transfer the upper phase (aqueous) to a fresh 1.5ml eppendorf and repeat the extraction
with 1 volume phenol/chloroform. Vortex and centrifuge as in step 3.
6.
Transfer the upper phase (aqueous) to a fresh 1.5ml eppendorf and add 1 vol chloroform.
Vortex for 30s and centrifuge for 2 min at max speed.
7.
Transfer the aqueous phase to a gDNA eliminator filter. Centrifuge at 10,000 rpm for 30
seconds.
8.
Add 600 μl ethanol (96-100%) to the lysate. Mix thoroughly by pipetting.
9.
Apply the sample to (approx 700μl) plus any precipitate to an RNeasy mini column in a
collection tube. Close gently, spin at 10,000rpm for 15 seconds. Discard flow-through.
Repeat this step if more sample remains, until all the sample has been added to the filter.
10.
Pipette 700 μl buffer RW1 into the RNeasy mini column, centrifuge for 15 seconds at
10,000 rpm (wash step). Discard flow-through.
11.
Pipette 500μl Buffer RPE onto the RNeasy column. Close lid gently, and spin at 10,000
rpm for 15 seconds. Discard flow-through.
12.
Add another 500μl Buffer RPE onto the RNeasy column. Close lid gently, spin at 10,000
rpm for 2 minutes (to dry membrane),
*optional: place RNeasy column in a new collection tube spin for a further minute at 10,000 rpm.
13.
Elution: transfer the column into a new collection tube (1.5ml). Pipette 30-50μl RNase free
water directly onto the silica-gel membrane. Close lid gently, spin at 10,000 rpm for 1
minute to elute. Do not discard flow-through.
14.
If greater than 30μg RNA yield is expected, repeat the elution as described with a second
volume of RNase free water into the same collection tube.
Check the quantity of RNA using the NanoDrop ND-1000. Need >1ug/ul and 15ug of RNA for the
labelling reaction