notes - Zymo Research HostZero Microbial DNA Kit (used for shotgun sequencing) - 16S error correction tools: > Divisive Amplicon Denoising Algorithm 2 or DADA2, an open-source R package that improves on the DADA algorithm > Quantitative Insights Into Microbial Ecology or QIIME (canonically pronounced 'Chime') - 16s bioinformatic tool: PICRUSt (pronounced “pie crust”) or Phylogenetic Investigation of Communities by Reconstruction of Unobserved States, written in Python and R Thanks for this wonderful video. 🌞
For the initial thawing step prior to adding DNA, it usually only takes about 2-3 minutes. We usually recommend flicking the tube with the competent cells just to see if the contents is still solid. If it is, then you can let the tube thaw for a few more minutes.
If I want to measure Methylation of protocadherin 17 human gene in gastric cancer cells before and after adding of dnmt1 inhibitor what is methods can be used ?
Absolutely! For samples stored in DNA/RNA Shield: - DNA: Stable for up to 2 years at room temperature - RNA: Stable for up to 1 month at room temperature For longer term storage, you can also store the samples at -20C or -80C.
Hi Habeebah, we would not recommend using a Thermo Mixer for the bead beating step. The Thermo Mixer will just shake the beads, but it won't move them in the right orientation so that they break open the microbial cells. In this case, you most likely will end up with low yields and underrepresentation of harder-to-lyse microbes like gram-positive bacteria or fungi. Here is a list of bead beating instruments and conditions that we would recommend: files.zymoresearch.com/documents/bead_beating_short_protocol_tables.pdf The easiest to set up would be the Vortex Genie from Scientific Industries with a Horizontal Tube Adapter (see links below): www.zymoresearch.com/products/vortex-genie-2 www.zymoresearch.com/products/horizontal-microtube-holder
Tip to increase plasmid yield exponentially: Before using kit, spin down 1mL of culture and discard supernatant. Re-suspend in 1:1 LB and H20, ~600uL. Continue using plasmic miniprep kit as instructed. This technique has increased yield 7x in some cases. Hope this helps someone else!
Thank you for sharing this tip. Sorry if this is a silly question, but why do you recommend resuspending in 1:1 LB and H2O specifically, versus just ddH2O or LB for example?
Increase NAD, decrease CD38 and control body inflammation seems to be the way right now to live a long healthy life that the average person can do right now.
Hi Maxine, you can use the PowerLyzer 24 Homogenizer for the bead beating steps. I believe that instrument is very similar to the Omni Bead Ruptor Elite instrument, so you can use those conditions as we've outlined here in our Optimized Lysis Protocols sheet files.zymoresearch.com/documents/bead_beating_short_protocol_tables.pdf:
Hey Zymo Research... pls do you have in store some references discussing about metagenomics vs metabarcoding to share with us (me especially)? Thanks by advance!
Good question! At this step, 95-100% ethanol can be used and the kit performance/RNA yield will be the same. The more important thing is that the ratio remains at 1:1 with the volume of TRIzol.
Yes, there is an appendix procedure for isolating plasmid DNA from Gram-Positive bacteria using the ZymoPURE Plasmid kits. You can find it on pg. 9 of the following Maxiprep protocol (link below). The main modification is to include a lytic enzyme (e.g. lysozyme) to help break down the cell wall of hardier Gram-Positive bacteria. files.zymoresearch.com/protocols/_d4202_d4203_zymopure_ii_plasmid_maxiprep.pdf
BS sales spiel. Tested negative with rapid and PCR, two days later at an airport tested positive. Came back to retest with a negative. How about a statistical report on how testing sites are following protocols…
I believe I've created an epigenetic emotional and mental psychological tool that's getting 100% results in resolving the cyclic negative behavioural patterns. I built it based on realising you have to work within a bloodline to be sure you get the root. Although I've got hundreds of real people case studies, I don't have anything scientific. I think this is a mahout missing live of the puzzle and it's organic. I also don't have the funding for an official study. Although my tool is now used in 20 countries by therapists. It really is a one of a kind. I think all studies need to also be segmented into males and females. Keep up the great work guys.
Great video. I have a question. In the first scenario it was around a 9% chance of a false positive and an almost 0% chance of a false negative. Do labs do any additional calculations to combine the two (positives and negative numbers) to give a more accurate result? Basically I’m trying to work out in the first scenario if the person got a positive result then does that mean it’s 91% accurate or is it more accurate than that when you factor in the negative numbers?? Hope that makes sense…
Hi, Thank you for your inquiry. Our Technical Support team would be more than happy to assist you or answer any of your questions, please send us an email to tech@zymoresearch.com and someone will get back to you shortly.
Hello, the strong fumes are usually from the TRIzol reagent. In low amounts, the fumes are not harmful. However, we recommend performing the sample lysis and loading step in a fume hood to minimize the amount of exposure to the fumes.
Great video! A suggestion for something you could do in the future might be to make a video comparing Amplicon Sequencing and Shotgun Sequencing, as 16S is just an example of an amplicon. You state that the use of 16S region limits your study to just bacteria, but other highly conserved regions can be used to target members of the other domains, like the 18S and ITS regions like you mentioned at the end.
Thanks for the video Doc :) A good explanation. I have a question though. From what I understand, at the current moment... we cannot know whether someone has covid-19 without doing the RT-PCR test, right? ... One can only know whether they are infected only based on this test. The thing is... when people talk about FP and FN (false positives and negatives) ... one thing that is required is the actual number of positive (true positive) people and also the actual number of those without the disease (true negative).... Now... since we don't have any true positive numbers (since the RT-PCR itself is not a gold standard)... how can FP or any other metric (e.g. sensitivity, specificity, or even FN) be reported or calculated? Ideally (or supposedly), before the RT-PCR would even be administered... one would have to have a sure way of knowing whether a person has the disease or not. Say this is done through isolating and purifying test subjects samples and absolutely determining whether or not he/she has the virus. In short, this is the gold standard... where the ground truth is known (meaning we know who actually has the virus, and who does not have the virus). Then, after these subjects have been properly classified.... then we run the RT-PCR on them... to see which patients are classified as +ve and which as -ve. .... and only then can we calculate the false positive (or whatever other) metric, right? Now... with the absenece of such subjects... how can we say that the RT-PCR has a FP of such and such? Or an FN of such and such....? Please note that this is not a negative comment. I am just trying to clarify based on what I've learned in my research on how to calculate false positives, sensitivity etc. :) Thank you!
if you're talking about Covid-19 specifically, the PCR tests according to the CDC, identify coronaviruses, not necessarily covid-19. This could lead to a quite high false positive rate.
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Could you introduce how to analyze differential methylation when you are free? And the theoroy about how to get methylation signal as well as interpret it in detail .
This doesn't explain the tests ability to recognize any coronavirus antibodies that have resulted from The Common Cold. You need to add that into your data to increase the odds of a false positive including a biological aspect. See CDC website regarding.
Curious how cheap I could drive sequencing for some very low complexity population samples. I'm interested in getting full genomes for some samples that probably have 10 or less species/strains as cheap as possible.