When running RNA on a Urea-PAGE gel, how would you differentiate between degraded RNA from non-denatured RNA? i.e., does a smear imply degradation/overloading and sharp non-specific bands imply secondary structures?
Hello folks, I found that my E.coli BL21 DE3 star cultures grew to O.D. 1.2 before I could induce with 0.5 mM IPTG. I normally induce at O.D. between 0.4 to 0.8 when they are in exponential...
07 August 2018 5,793 0 View
Hello folks, I had a N terminal His-GST-TEV cleavage site tagged protein (15mg) on dialysis over ~40 hours in the presence of TEV protease (1mg) with a buffer change in between. Buffer is 50 mM...
06 July 2018 1,320 5 View
Hello folks, I am performing Ni-NTA purification of N-term His-GST-TEV cleavage site -tagged protein. Due to a time limitation I need to leave it overnight in wash buffer (NaH2PO4 pH 8.0, 300mM...
06 July 2018 4,878 4 View
Hello there, I have prepared 10-55% sucrose gradients containing 1mM Chloramphenicol and run them on an Isco sucrose gradient fractionation system, but the absorption is too high to record on the...
03 April 2016 9,905 3 View
Hello, I am currently having problems with RNA extraction. I am using mouse liver (C57BL6J), and I have extracted RNA from mouse liver before. Before this experiment, my final RNA pellets were...
11 August 2024 7,082 3 View
I got these smeared bands quite often lately. We typically run the gel at 140V with a 10-12% gel and do a wet transfer at 220 mA for 1.5 hr in cold room. We also noticed some dirty spots/dots (see...
10 August 2024 7,480 3 View
I have been doing the m6A dot blot for a while with no improvement, I am extracting the RNA, and I can see the dots although the three biological replicas give a different reading on the memberan...
10 August 2024 8,539 5 View
I've been performing RNA extraction on cotton petiole tissue for a few months now using the method described in the following paper, a derivative of the typical hot borate method...
08 August 2024 9,882 2 View
I am currently working on LncRNA; to know the lncRNA-protein interactions I want to do RNA pull down assay, so I need to design primers with T7 promoter. I need assistance in this regard.
07 August 2024 6,622 1 View
Dear QE-users, In the method where full MS positive mode and PRM mode are used, we always get an incorrect auxiliary gas reading (41 instead of 25). This only happens in this method; other...
06 August 2024 4,953 0 View
Recently, we observed that 99% of the sequences in our RNA-seq data corresponded to the E. coli genome. Despite multiple DNAse treatments after RNA extraction and ribosomal depletion, we were...
06 August 2024 807 3 View
I am planning to collect human fecal samples for metatranscriptomic analysis using MGI. These samples are from indigenous people living in a region with high temperatures. I will have access to a...
06 August 2024 1,367 3 View
I have been working on Red blood cell-derived extracellular vesicles as Antisense Oligonucleotide (ASO) carriers. We normally run agarose gel to quantify the loading efficiency. I used naked ASO...
06 August 2024 3,130 2 View
I am having an issue with my gel image where my PCR product is not appearing very bright on the gel. When I perform gel extraction, the A260/280 purity value is very low. I used the Qiagen gel...
05 August 2024 9,798 3 View