Cell lysate: Will high heat inactivate all proteases?

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...

02 March 2020 5,957 3 View

QPCR: Manually adjusting threshold value for Ct?

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 =...

01 February 2020 3,833 3 View

qPCR: Minimal Ct for accuracy?

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...

01 February 2020 6,654 4 View

Safe RCF/g/centrifuge speed for enzymes and other proteins?

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...

01 February 2020 7,260 5 View

qPCR melt curve: One sample in standard curve had shifted peak?

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....

01 February 2020 8,392 3 View

Dilution factor necessary for log phase cultures?

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...

31 December 2019 9,056 8 View

TRIzol RNA extraction: No pellet after isopropanol precipitation even with glycogen?

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....

31 December 2019 3,856 5 View

Bacterial electroporation: Recovery media volume and duration of recovery?

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,...

31 December 2019 6,237 3 View

Periodic low yield/degradation of RNA?

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...

31 December 2019 4,191 2 View

Precipitation of RNA pellet with isopropanol: Spread/smear of pellet across side of tube?

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...

31 December 2019 1,859 13 View

Gel electrophoresis result for total RNA samples, 1.5 Agarose gel was used, How to improve the integrity of RNA?

Gel electrophoresis, RNA degradation, RNA extraction from fresh tissue

02 March 2021 5,433 5 View

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02 March 2021 3,060 3 View

Issues with Permeabilization during Flow Cytometry?

Hello Everyone. Currently I am working to characterize macrophages in the myocardium after ischemia-reperfusion injury in rats. Due to the low total cell number isolated from rat hearts I can...

01 March 2021 3,867 3 View

Which cell line should we use to perform biological dosimetry experiments?

We are preparing some experiments based on irradiating cells under different conditions in order to evaluate the effects in terms of DNA damage, genetic expression, etc. As our project is...

01 March 2021 3,355 3 View

Is it possible for two human neuronal cell lines to have different gene sequence for the same protein?

I am looking at the ATP1A2 (Sodium/Potassium ATPase alpha subunit 2) in two human neuronal cell lines. Expression levels of this protein seems to be almost equal when detected by one antibody....

01 March 2021 3,607 3 View

Is CD44 degraded in lysosome together with hyaluronan after CD44 mediated endocytosis of hyaluronic acid?

Also when RHAMM binds hyaluronic acid, they get internalized, will RHAMM also be degraded? Or both CD44 and RHAMM will be transferred back to the cell membrane? Asking for breast cancer cell line...

01 March 2021 8,169 2 View

Do you need the CMV enhancer before the CMV promotor for a proper function of CMV on the gene that follows?

I'm a student and I have to produce a cell line with a knockin for the NRF2 gene with GFP. I have to put a promotor in front of the GFP because the gene will be too far away from the promotor of...

28 February 2021 7,127 2 View

Problem with shRNA transfection. Why are only two plates getting contaminated instead of all?

I transfected my LNCaP-WT cells with 3 shRNA plus their NTC two weeks ago and split two puromycin selected cell plates on Friday last week(Feb 26). I checked for GFP in the cells, and they all...

28 February 2021 4,949 3 View

What could be some reasons why unstimulated monocytes are getting activated?

Im doing PBMC isolation -> CD14+ enrichment using magnetic beads -> stimulation setup. My negative control is just cells in cRPMI but they seem to get activated over and over again.

28 February 2021 7,883 3 View

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28 February 2021 9,936 2 View