Biology
A subscription to JoVE is required to view this content.
You will only be able to see the first 2 minutes.
The JoVE video player is compatible with HTML5 and Adobe Flash. Older browsers that do not support HTML5 and the H.264 video codec will still use a Flash-based video player. We recommend downloading the newest version of Flash here, but we support all versions 10 and above.
If that doesn't help, please let us know.
Microbiota Analysis Using Two-step PCR and Next-generation 16S rRNA Gene Sequencing
Chapters
Summary October 15th, 2019
Described here is a simplified standard operating procedure for microbiome profiling using 16S rRNA metagenomic sequencing and analysis using freely available tools. This protocol will help researchers who are new to the microbiome field as well as those requiring updates on methods to achieve bacterial profiling at a higher resolution.
Transcript
Profiling of microbiome is the first step toward defining the role of microbiome in health and disease.Here, we are describing a simple procedure for isolating high-quality bacterial DNA from fecal sample preparing library, performing 16SR RNA and a meta genomic sequencing and microbiome data analysis.This protocol will help researchers new to microbiome field, as well as researchers working in microbiome field in establishing the microbiome pipeline in their lab.This protocol can be extended for profiling of microbiome from other bio specimens such as nasal, oral, or skin samples of animal and human subjects.Bead picking is the most important steps as all downstream steps are depend on high quality DNA.Proper sealing of preserve plate is important as incomplete sealing might lead to the oppressing of amplified product resulting in low quality sequencing data.To begin, sterilize the divider boxes with 70%ethanol.Place the mice in sterilized divider boxes and allow them to defecate normally for up to one hour.Use sterile forceps to collect the fecal pellets in an empty pre-labeled 1.5 milliliter micro centrifuge tube.Enclose the tube securely.Sterilize the forceps with 70%ethanol, followed by a germicidal disposable wipe, after collecting fecal from each mouse.Weigh 200 milligrams of fecal pellets into a 1.5 milliliter micro centrifuge tube, with 0.1 milliliter glass beads, and place the bead tube into a bead beating homogenizer and homogenize the samples for 45 seconds at room temperature, at a speed of 4.5 meters per second.Extract DNA as per instructions of the kit manufacturer and elute DNA in a 1.5 milliliter micro centrifuge tube.After isolating DNA, quantify the isolated DNA by loading 1 microliter of the DNA on a fluorometer.Setup 16S ribosomal RNA gene amplification PCR1 in a 96-Well PCR plate, using a 25 microliter reaction volume.Using a multi-channel pipette, add 40 nano grams of DNA, 13.5 micro liters of 2X high-fidelity polymerase enzyme mix, containing buffer, plus DNTPs in forward and reverse primer.Seal the PCR plate properly from the top to the bottom in along all four edges.And centrifuge at 1000 times G at 20
Related Videos
You might already have access to this content!
Please enter your Institution or Company email below to check.
has access to
Please create a free JoVE account to get access
Login to access JoVE
Please login to your JoVE account to get access
We use/store this info to ensure you have proper access and that your account is secure. We may use this info to send you notifications about your account, your institutional access, and/or other related products. To learn more about our GDPR policies click here.
If you want more info regarding data storage, please contact gdpr@jove.com.
Please enter your email address so we may send you a link to reset your password.
We use/store this info to ensure you have proper access and that your account is secure. We may use this info to send you notifications about your account, your institutional access, and/or other related products. To learn more about our GDPR policies click here.
If you want more info regarding data storage, please contact gdpr@jove.com.
Your JoVE Unlimited Free Trial
Fill the form to request your free trial.
We use/store this info to ensure you have proper access and that your account is secure. We may use this info to send you notifications about your account, your institutional access, and/or other related products. To learn more about our GDPR policies click here.
If you want more info regarding data storage, please contact gdpr@jove.com.
Thank You!
A JoVE representative will be in touch with you shortly.
Thank You!
You have already requested a trial and a JoVE representative will be in touch with you shortly. If you need immediate assistance, please email us at subscriptions@jove.com.
Thank You!
Please enjoy a free 2-hour trial. In order to begin, please login.
Thank You!
You have unlocked a 2-hour free trial now. All JoVE videos and articles can be accessed for free.
To get started, a verification email has been sent to email@institution.com. Please follow the link in the email to activate your free trial account. If you do not see the message in your inbox, please check your "Spam" folder.
