"b -mercaptoethanol is used to help to destroy RNases that may be present and will degrade the RNA. b -mercaptoethanol is a reducing agent that will reduce the disulfide bonds of the RNases, thereby destroying the conformation and the functionality of the enzyme". It comes from http://www.norgenbiotek.com/index.php?id=faqs_rnakits
Seventy percent ethanol is commonly used in RNA extraction to wash and remove salts and contaminants from the RNA sample. It helps to purify the RNA by precipitating it out of the solution while leaving behind impurities. Additionally, the 70% ethanol concentration helps minimize RNA degradation during the extraction process.
I have not personally used the Qiagen Total RNA Extraction Kit for RNA extraction.
Adjusting the pH to 7 during RNA extraction helps to create the optimal conditions for RNA stability. RNA is more stable at a neutral pH, which minimizes degradation and helps maintain the integrity of the RNA molecules during the extraction process. This ensures that high-quality RNA is obtained for downstream applications.
QIAzol Lysis Reagent is used to lyse cells and tissues to release RNA for extraction. It disrupts the cellular and nuclear membranes, thus allowing the RNA to be isolated and purified from the lysate.
BCP bromo chloropropane is commonly used as a solvent for RNA isolation to disrupt cell membranes, denature proteins, and protect RNA from degradation. It helps to separate RNA from other cellular components during the extraction process, making it easier to isolate pure RNA for downstream applications such as reverse transcription and gene expression analysis.
Seventy percent ethanol is commonly used in RNA extraction to wash and remove salts and contaminants from the RNA sample. It helps to purify the RNA by precipitating it out of the solution while leaving behind impurities. Additionally, the 70% ethanol concentration helps minimize RNA degradation during the extraction process.
75% ethanol is commonly used in RNA extraction because it helps to wash the RNA pellet by removing salts and other contaminants, while also helping to maintain the integrity and stability of RNA molecules. The lower ethanol concentration reduces the risk of RNA degradation and allows for efficient RNA recovery during the extraction process.
This wash step allows you to centrifuge the sample and collect a "clean" RNA pellet, after discarding the supernatant that contained contaminating salts and proteins. When isolating and purifying RNA, 75% ethanol is used as a wash solution because RNA is a precipitate (solid) in this percentage of ethanol, while most proteins and salts remain in solution (are soluble). At a lower % ethanol, both the RNA and the proteins would be soluble, so you would not be able to separate them. At a higher % ethanol, both the RNA and salts would remain in the pellet, so you would not be able to separate the salts from your RNA. Prior to the wash step, you probably added 100% ethanol to your sample, so the final total concentration of ethanol was 75%. This step is where the RNA precipitates out of solution. You would then centrifuge the sample and discard the supernatant, as above. In the wash step, you are merely using the same solution (75% ethanol) to wash the RNA pellet you created in the previous step.
I have not personally used the Qiagen Total RNA Extraction Kit for RNA extraction.
Chloroform is commonly used in RNA extraction to separate RNA from other cellular components. It helps in the denaturation of proteins and the dissolution of lipids during the extraction process. Chloroform aids in the formation of a distinct organic phase where RNA can be collected.
RNAse destroys the RNA and hence RNAse contamination is a problem in RNA extraction as it breaks down RNA. RNAse enzyme is removed by using RNAse inhibitor or precautions like wearing of gloves, autoclaving tips , using RNAse free water/DEPC treated water is done while performing RTPCR
TE stands for Tris and EDTA. The Tris buffers the water to prevent acid hydrolysis of the DNA/RNA. The EDTA chelates divalent cations that can assist in the degradation of RNA.
Adjusting the pH to 7 during RNA extraction helps to create the optimal conditions for RNA stability. RNA is more stable at a neutral pH, which minimizes degradation and helps maintain the integrity of the RNA molecules during the extraction process. This ensures that high-quality RNA is obtained for downstream applications.
Ethanol is commonly used in microbiology labs as a disinfectant to sterilize surfaces, equipment, and lab benches. It is also used for flame sterilization of inoculating loops and needles. Additionally, ethanol is used in DNA and RNA extraction protocols to precipitate nucleic acids.
QIAzol Lysis Reagent is used to lyse cells and tissues to release RNA for extraction. It disrupts the cellular and nuclear membranes, thus allowing the RNA to be isolated and purified from the lysate.
Isopropanol is used in RNA extraction to precipitate RNA from the sample solution. By adding isopropanol to the sample, RNA molecules clump together and can be separated from the rest of the components in the solution using centrifugation. This allows for the isolation of RNA for further analysis.
In a DNA extraction, the purpose of a buffer is to solubilize DNA as well as RNA. Because of this, it prevents the DNA for degrading.