promptness is key to obtain high-quality RNAs, we avoid this
especially when extracting RNAs for RNA-Seq.
13. This step should be performed under a fume hood as chloroform is highly volatile and toxic.
14. In order to prevent contamination with DNA and precipitated
proteins, great care should be taken to avoid perturbating the
three phases. It is better not to try to retrieve the totality of the
upper phase to prevent carryover.
15. You can pause at this step, and store the samples in isopropanol
at À20
C until you are ready to proceed with the procedure,
although we avoid this especially when extracting RNAs for
RNA-Seq.
16. You should be able to see a white gel-like pellet containing
RNA at the bottom of the tube. Great care should be taken
when removing the supernatant as sometimes the RNA pellet
does not tightly stick to the tube and tends to move on the
tube wall.
17. You can pause at this step, and store the samples in 75% ethanol
at À20
C until you are ready to proceed with the procedure,
although we avoid this especially when extracting RNAs for
RNA-Seq.
18. As washing the RNA pellet with 75% ethanol will dissolve salts
contained in the pellet, the aspect of the pellet will change.
Very often, the pellet becomes smaller and less visible. Again,
great care should be taken when removing the supernatant.
You can use a micropipette or a syringe with a 22 G needle to
remove most of the supernatant without disturbing the pellet.
19. The time for air-drying the RNA pellet will depend on the
amount of ethanol left in the tube after removing the supernatant. You can put your samples under a fume hood or laminar
follow cabinet to enhance the drying step. If the ethanol is
properly removed, this step should take 5–10 min. You should
avoid excessively drying the RNA pellet as it will decrease its
solubility.
20. You should use a clean water bath. A heat block might work
as well.
21. To enhance the DNase reaction, you can perform the reaction
with 2 μl (4 U) of Turbo DNA-free™ DNase. You can also
perform a two-step incubation with the enzyme. In a first step,
1 μl of Turbo DNA-free™ DNase are added and the sample is
incubated for 30 min at 37
C. Then, an additional 1 μl of
Turbo DNA-free™ DNase is added to the sample, and the
second incubation at 37
C is conducted for 30 min.
22. The amount of DNase Inactivating Reagent to be added
should be adjusted depending on the number of DNase units
Total RNA Extraction from Leptospira
49
especially when extracting RNAs for RNA-Seq.
13. This step should be performed under a fume hood as chloroform is highly volatile and toxic.
14. In order to prevent contamination with DNA and precipitated
proteins, great care should be taken to avoid perturbating the
three phases. It is better not to try to retrieve the totality of the
upper phase to prevent carryover.
15. You can pause at this step, and store the samples in isopropanol
at À20
C until you are ready to proceed with the procedure,
although we avoid this especially when extracting RNAs for
RNA-Seq.
16. You should be able to see a white gel-like pellet containing
RNA at the bottom of the tube. Great care should be taken
when removing the supernatant as sometimes the RNA pellet
does not tightly stick to the tube and tends to move on the
tube wall.
17. You can pause at this step, and store the samples in 75% ethanol
at À20
C until you are ready to proceed with the procedure,
although we avoid this especially when extracting RNAs for
RNA-Seq.
18. As washing the RNA pellet with 75% ethanol will dissolve salts
contained in the pellet, the aspect of the pellet will change.
Very often, the pellet becomes smaller and less visible. Again,
great care should be taken when removing the supernatant.
You can use a micropipette or a syringe with a 22 G needle to
remove most of the supernatant without disturbing the pellet.
19. The time for air-drying the RNA pellet will depend on the
amount of ethanol left in the tube after removing the supernatant. You can put your samples under a fume hood or laminar
follow cabinet to enhance the drying step. If the ethanol is
properly removed, this step should take 5–10 min. You should
avoid excessively drying the RNA pellet as it will decrease its
solubility.
20. You should use a clean water bath. A heat block might work
as well.
21. To enhance the DNase reaction, you can perform the reaction
with 2 μl (4 U) of Turbo DNA-free™ DNase. You can also
perform a two-step incubation with the enzyme. In a first step,
1 μl of Turbo DNA-free™ DNase are added and the sample is
incubated for 30 min at 37
C. Then, an additional 1 μl of
Turbo DNA-free™ DNase is added to the sample, and the
second incubation at 37
C is conducted for 30 min.
22. The amount of DNase Inactivating Reagent to be added
should be adjusted depending on the number of DNase units
Total RNA Extraction from Leptospira
49