Is there any specific transcriptomics marker for different phases of cell cycle?

Can anyone please

I did the detailed manual annotation of certain lncRNA where I searched out Host, upstream, and downstream genes and the T-DNA insertion mutant available lncRNA chromosomal coordinates. Now my question is there any scientific way to present this annotation in a Research paper which may be a Figure, network, or else?

Could anyone suggest a protocol to determine the conc. of puromycin required to kill CHO-S cells? If anyone has performed earlier, can you please suggest a range of puromycin to be tested?

Formamide denature secondary structure of RNA, so no dsRNA should be present. Then how intercalation is possible

Hello everyone, I have the following problem: I'm observing a lot of smear after loading qPCR products on 1.3% gel (attached pUC19-M13_primers_test) eventhough the melt curves seem nice with only one peak (attached melt_curves_pUC19). In the reaction I've used 10-fold dilutions of pUC19 fragment (121 bp) using M13 primers (from 1E7 (St7) down to 1E4 (St3)). I've tried if different primer concentrations show any difference but to me it all looks the same. Standard curves of different primer concentrations showed between 89-92% efficiency. This was done on QuantStudio5 instrument, with 384 well block. So my questions are the following:

1. How come that only one peak can be observed on a melt curve while a smear of different products can be observed on a gel?

2. What is causing the smearing on a gel? If it would be the gel or any of the components of the electrophoresis probably also the ladder would be smeared or?

P.S. Primers were freshly prepared, the same goes for the plasmid dilutions, which were diluted from the commercial stock solution. Amount of DNA in the well with the highest DNA load was negligible (0.03 ng/uL) as calculted from the 1E7 copy number of pUC19 plasmid. Electrophoresis was run in 1xTAE buffer, the same buffer was used for preparing the gel.

Thank you very much in advance!

I have taken a water sample from point that pass trough two new water cartridge with filter 0.45 & 0.2 micron, so it's seem to be sterile !

and filtrated it with 0.45 Millipore filter, after incubation I found the sample is very crowded with colonies, knowing that the source is very crowded also with bacteria.

Does the high load of bacteria can pass throw the filter cartridges ?

AND WHY PASS throw the cartridge but not in the (0.45 Millipore filter)?

I’m currently trying to transfect LAN5 cells (human neuroblastoma cell line) using the Lipofectamine 3000 transfection reagent, both with the vector pCMV6-AC-GFP expressing the human heat shock 60kDa protein (Origene RG224428 vector) and with a control plasmid (the same vector without the insert, Origene PS101000 vector).

Using a DNA : Lipo ratio of 1:3, I obtained a good (but not high) transfection efficiency with the control plasmid, but no transfection, or a very low transfection efficiency with the expression plasmid.

Could the plasmid size be responsible for this different transfection efficiency?

Does anyone have any advice to improve the transfection efficiency for both plasmids?

Thanks for your suggestions!!!

Hi

Would you mind please explain the difference between assessing viability using Cell Counting Kit-8 (CCK-8) and MTT assay?

Thank you

Hello everyone. I'm having some issues with a qPCR that is intended for the amplification of bmoX gene.

I did a standard curve with a normal PCR product of bmoX, which was purified and then diluted.

In Rotorgene Software I apparently have a good efficiency and good R/R2 (imagen below), although I have primer dimers. But when I run the samples, after the melting curve, in agarose gel I see a huge smear all along the well in all 6 samples (even in the negative control, that has only water, primers and sybrgreen mix). I also included the standard curve in the agarose gel, to be sure it isnt degraded but I can never get a visible band. The only band I can see corresponds with the initial PCR product of bmoX (that is the stock of my standard curve)

The unknown sample is a soil DNA extraction. It doesnt necessarily has the gene.

Te mix consisted in:

10 uL SensiFAST SYBR No-ROX Kit

0.6 uL of each primer

5 uL DNA

and water up to 20 uL

Amplification program is:

95°C 5 minutes

40 cycles of:

95°C 45 sec

60°C 30 sec

72°C 60 sec

I hope the information was clear. I'm really struggling since I don't really know what is happening, and this happend many times now.

I also want to add that I was first using the Rotor-Gene SYBR Green PCR Kit, and I never had primer dimers, and I just recently changed the kit and now I have in every qPCR run.

I would really appreciate if you could help me out!!