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Questions related from Gen Gi
I'm extracting protein from highly hazardous bacterial samples. For samples like this, people commonly boil the bacteria prior to full lysis. Because the bacteria is boiled, do I still have to...
03 March 2020 6,075 3 View
Sometimes I need to centrifuge reactions containing enzymes, however, I've realized I have absolutely no idea if spinning very fast could damage the enzymes. Does anyone have any data on safe...
02 February 2020 7,413 5 View
To study gene expression with qPCR, I'm using 16s as a reference gene. 16s is of course very, very abundant so I need to dilute my cDNA a lot. However, I don't want to overdilute my cDNA as then...
02 February 2020 6,755 4 View
I serially diluted my cDNA to generate a standard curve. For the melt curve, all the samples in the standard had one, nice peak. Except one sample in the very middle of the dilution series....
02 February 2020 8,542 3 View
I'm using a CFX96 and when the machine automatically picks a Ct, I get very bad efficiency values (~250%). However, when I manually move the threshold for Ct much lower, I can get: r-squared =...
02 February 2020 3,980 3 View
Does anyone have/know any data on how the volume of recovery media affects things for electroporation of bacteria? I see large ranges. For electroporating, eg, 200 uL concentrated bacteria,...
01 January 2020 6,392 3 View
For my protocols, I'm supposed to electroporate at: 2.5 kV 1000 ohms for resistance 25 uF for capacitance However, some electroporators appear only to allow adjustment of the voltage. Does it...
01 January 2020 5,413 1 View
Many microbiology processes involve: 1. Growing a starter culture to high density 2. Back diluting into fresh media by some dilution factor, eg, 1:100 3. Growing to OD X (eg, OD=0.5) 4. Harvesting...
01 January 2020 9,174 8 View
Is it important to avoid the peripheral wells for qPCR? Our qPCR machine is only 96w, not 384w, so losing all the peripheral wells is extremely inconvenient, but of course I'd rather not...
01 January 2020 6,017 3 View
So I'm using TRIzol for RNA extraction, but suddenly I'm getting no pellet during the isopropanol step *even though I added glycogen.* A few weeks ago I: 1. Took a large quantity of bacteria 2....
01 January 2020 4,068 5 View
Many microcentrifuge tubes are designated as both "RNase-free" and "non-sterile." What does this mean exactly? I can't imagine something could truly be both RNase-free and non-sterile. Is it...
01 January 2020 5,809 3 View
So I want to DNase treat my RNA and inactivate the DNase. Then later synthesize cDNA and do qPCR The DNase inactivation is often done via heating 10-20 min at 65-75C. However, cations can...
01 January 2020 8,533 2 View
I'm extracting RNA from bacteria and I only get a yield ~20% of the time. I can take one culture, split it into multiple tubes, prep them in parallel, and only one tube will give me RNA. I...
01 January 2020 4,298 2 View
When precipitating RNA pellets with isopropanol, the RNA pellet is sometimes "smeared" across the side of the tube instead of being one small pellet (after centrifuging that is). It's still very...
01 January 2020 1,998 13 View
My RNA 260/280 is 1.9 and the 260/230 is 2.0. Is that good enough for qPCR and RNAseq? Or should I optimize the purification? What's the lowest the ratios can be?
12 December 2019 9,422 4 View
I'm using TRIzol+bead beating to extract RNA from a gram positive bacteria, but I'm getting very low yields (
12 December 2019 7,363 3 View
A lot of protocols say to avoid washing cells with PBS before addition of TRIzol as this increases the chance of RNA degradation. An example is the official TRIzol documentation:...
12 December 2019 8,116 3 View
Months ago I used specific primers for lots of genotpying PCR and they always worked beautifully with taq polymerase. Yesterday I did the PCR with those same primers that worked so well: No...
11 November 2019 3,425 14 View
For TURBO DNase, is buying the full kit that uses bead-mediated removal of the DNase worth it? Or can I just buy the TURBO DNase itself and use heat inactivation? I'm mainly interested in using...
11 November 2019 7,011 1 View
