RNA isolation / purification Bacteria - Gram positive Bacillus anthracis

The biggest problem in isolating RNA from gram-positive bacteria is the disruption of the cell wall. A lot of protocols employ enzymatic digestion (pretreatment) which may affect gene expression patterns of certain genes. Therefore physical disruption using beads can be a best alternative.

3 Matching solutions
1 Discussion

Found 1 discussion for this experiment

Discussion

5 years ago

5 years ago by Paul G. Macon United States

Some help with RNA isolation using Trizol

Hello! I used Trizol to extract total RNA from in-vitro cultured bacteria (1 X 10^8 cells). After phase separation, I mixed ~0.4 ml of the upper phase which contains RNA with 0.5 mL cold isopropanol. However, the amount of RNA when measured in Nanodrop was very low. In addition, the ratio between 260 and 230 was around 0.1 to 0.5. Is there a chance that my sample was contaminated by the Trizol reagent? When I collected the aqueous phase I made sure to not touch the lower phase. What should I do?

Comment View 4 comments

Share your thoughts or question with experts in your field by adding a discussion!

Found 3 matching solutions for this experiment

AllPrep DNA/RNA Mini Kit

Qiagen

Upstream tips	Protocol tips	Downstream tips
Complete disruption of plasma membranes of cells and organelles is absolutely required to release all the nucleic acids contained in the sample. Different samples require different methods to achieve complete disruption. Incomplete disruption results in significantly reduced nucleic acid yields. Overloading the spin columns significantly reduces nucleic acid yields.	Homogenizing the material is necessary to redice the viscosity of the lysates caused by cell disruption. Incomplete homogenization results in inefficient binding of DNA and RNA and therefore significantly reduced yield and purity of nucleic acids. Excessive homogenization, on the other hand, results in shorter genomic DNA fragments. The centrifugation temperature should be 20–25ºC. Warm the lysate to 37ºC before transferring it to the AllPrep DNA spin column.

Upstream tips
Complete disruption of plasma membranes of cells and organelles is absolutely required to release all the nucleic acids contained in the sample. Different samples require different methods to achieve complete disruption. Incomplete disruption results in significantly reduced nucleic acid yields. Overloading the spin columns significantly reduces nucleic acid yields.
Protocol tips
Homogenizing the material is necessary to redice the viscosity of the lysates caused by cell disruption. Incomplete homogenization results in inefficient binding of DNA and RNA and therefore significantly reduced yield and purity of nucleic acids. Excessive homogenization, on the other hand, results in shorter genomic DNA fragments. The centrifugation temperature should be 20–25ºC. Warm the lysate to 37ºC before transferring it to the AllPrep DNA spin column.

Manufacturer protocol Publication protocol 1 Relevant paper

RNeasy Mini Kit

Qiagen

Upstream tips	Protocol tips	Downstream tips
	To yield high RNA, give the column an extra wash with both RW1 and RPE buffers (3 total washes with each buffer).	"Include DNAse treatment for 15-20min. Ensure EtOH is completely evaporated off of the column prior to elution. Adjust time from 1min to 5 min at 60`C. Use water to elute the RNA that is warmed to ~60`C"

Protocol tips
To yield high RNA, give the column an extra wash with both RW1 and RPE buffers (3 total washes with each buffer).
Downstream tips
"Include DNAse treatment for 15-20min. Ensure EtOH is completely evaporated off of the column prior to elution. Adjust time from 1min to 5 min at 60`C. Use water to elute the RNA that is warmed to ~60`C"

Manufacturer protocol Publication protocol 1 Relevant paper

RNeasy Lipid Tissue Mini Kit

Qiagen

Upstream tips	Protocol tips	Downstream tips
	If the RNA yield is low, repeat RNA elution, but incubate the RNeasy Mini spin column on the benchtop for 10 min with RNase-free water before centrifuging. All centrifugation steps should be performed at 15–25°C except for phase separation

Protocol tips
If the RNA yield is low, repeat RNA elution, but incubate the RNeasy Mini spin column on the benchtop for 10 min with RNase-free water before centrifuging. All centrifugation steps should be performed at 15–25°C except for phase separation

Manufacturer protocol Publication protocol 1 Relevant paper

Can't find the product you've used to perform this experiment? It would be great if you can help us by Adding a product!

Outsource your experiment

Fill out your contact details and receive price quotes in your Inbox

Outsource experiment