Do ~300bp gene fragments melt at boiling temperatures?

I would like to clone a large (~40 kb) plasmid.

I am wondering if I should use a high copy or low copy backbone for the plasmid?

I would much prefer high copy number to get greater DNA yield and purity, but I have heard low copy greatly improves stability of large plasmids.

Is using low copy number important for large plasmids?

I'm passionate in oncology research. I did bachelor in Zoology and now pursuing MPhil Molecular Biology and Biotechnology and working on cancer genetics. I am greatly interested to do PhD in cancer research from a world renowned institute but I think with this profile I would get a position in top ranked institute for PhD. Should I go for another master from a renowned foreign institute with major in oncology?

Thanks

Hello everyone,

It is my understanding that polyA signal isn’t absolutely necessary for mammalian gene expression but it’s highly recommended to use it whenever possible. I’m wondering if lack of such signal can lead to the complete absence of the protein expression. The reason why I’m asking this is because I work with the construct that is used for transgenesis through recombination. On this construct I have a Tet-dependant promoter that I know works and my gene of interest downstream of it, but there is no polyA signal. After recombination of this cassette into the genome of the cells that already express rtTA and culturing in the presence of dox, I was hoping to see the expression of my gene of interest. I could indeed detect mRNA expression by qPCR (there was a significant difference between wt (non-recombined cells), -dox and +dox conditions; Cts for +dox were still relatively high though). However, when I performed IF with the antibody that I know works (it worked for the positive control) I couldn’t detect my protein. I also performed a functional test, from which it was clear that I don’t have any functional protein in the recombined cells. I sequenced the whole plasmid that I used for recombination as well DNA from the cells after recombination and the sequences seem to be completely correct. Since I can detect mRNA, I was wondering if it’s possible that my construct gets transcribed (although probably with an extensive 3’ UTR) but can’t be properly/efficiently translated due to the lack of PolyA signal. Or is there any better explanation for the absence of the protein

may I ask about if there is any human gene expression only depends on core promoter,which no need upstream any enhance and regulator ?

As I am wondering, if there are some gene, they are in modulation function, which also they are in very upstream of signal for a cell, therefore their core promoter 's binding affinity is strong enough stablizing the transcription complex, and so no need enhancer and upstream regulatory protein .Thank you so much

Hello, I'm preparing libraries for Illumina sequencing and i would like to know if I have to do DNA end repair immediately after shearing, or if I can freeze sheared DNA and do end repair a week or two later. Thanks

I am analyzing some western blots that I had to strip and re-probe for the house-keeping gene loading control. For the quantification, I'm using the ImageLab software from Bio-Rad. I'm concerned that if I use the value from the stripped/reprobed loading control for normalizing the sample values, it won't be accurate because the "background" signal won't be the same for both due to the stripping process. Is this a valid issue?

Goodnight

Sorry to bother in this Sunday

But I would like to ask a few questions:

Does anyone know about how to induce the generation of polyploid giant cancer cells (PGCCs) in melanomas with "targeted drugs"?

How to measure the presence of polyploidy in the tumor?

How to induce the generation of giant polyploid cancer cells (PGCCs) in melanomas?

Is there any protocol to induce the generation of polyploid giant cancer cells (PGCCs) in melanomas?

Is there any way to study the immortalization and regeneration of tumors with cellular and molecular biology? Is there any protocol?

Best regards,

Matheus Correia Casotti

Does the DNA remain stable or degrade at this temperature? Would there be any difference in thermal stability between supercoiled and linear forms of say, 3 kb plasmid.

Hello everyone,

I hope this message finds you doing well.

​I am writing to ask you a significant question about whole-virus ELISA and its procedure.

Honestly, it seems that coating a microplate with viruses is not as convenient as some papers mentioned, especially when high accuracy is needed. Now, my question is, is there any particular procedure in order to enhance the efficiency of the coating? For instance, what would we do if we tended to expose viral protein to the microplate better than before, based on your experience?

Thank you