Bump. I am also interested in finding out how they do it. I mean actually, not guesswork.
- → CPRES's Content
There have been 167 items by CPRES (Search limited from 17-April 20)
Yes, you can transfer from -40 to LN2 without problem. I have done it several times with several cell lines, out of impatience, so I am sure it works.
However, don't do it with some sensitive or irreplaceable cell line without doing some trial runs. And if you do, don't sue me. What's with your Avatar name by the way?
gene expression is controlled by several things:
1. Transcription factors (repressors and activators)
2. DNA epigenetic changes
3. Histone epigenetic changes
4. Enhancers (long-range)
In addition, once a genomic transcript is generated, it can be spliced, truncated, degraded in so many ways that it changes eventual protein levels. Also, protein can be controlled in several ways (ribosomal processing, tagging for degradation etc) during and after translation.
And this is very basic idea, there is a lifetime of complexity to talk about here.
No, here is what you do:
1. Buy a siRNA that is targeting your gene. This is unlabeled siRNA directed against your gene.
2. Buy a control (BlockiT) alexa fluor red siRNA. This siRNA is carefully designed such that it does not target any mammalian gene. So, it is a negative control, but more importantly, it is a transfection efficiency control.
And that is all you need. Forget about a kit to label your siRNA, unless you want to see its cellular localization and dynamic. Most experiments are just about transfecting siRNA to knockdown the gene of your interest.
The BLOCK-iT Alexa Fluor Red is already labeled siRNA, so you don't have to label or incorporate anything (if that is what you are concerned about).
Just use this fluorescent siRNA exactly as you use your experimental siRNA in the protocol.
There is nothing to it. You will be able to see if you are getting a good transfection efficiency with your method.
Basically, you transfect your experimental siRNA and control (fl labeled) siRNA to your cells (in separate wells of course). Then check the control cells under fl microscope to see what % of cells show fl.
Labnerd: "However, I feel like if we submit the manuscript to this journal, it will bypass the editorial board and head straight to the reviewers, which I think will pretty much guarantee an acceptance."
This is precisely you should not submit to the journal. Just as you think you can bypass the process, other people will think you probably bypassed the process and paper was otherwise not much worth. Go with your supervisor's advice. Believe in your work.
Hi Funny Fish, I don't want to discourage you, but I have attempted such thing and has never worked for me. But who knows it might work for you.
If you have enough unstained sections with you, it is always best to do a clean experiment.
You got to do some work, man!
It took me 30 seconds to view your photograph, and 30 seconds to do google image search (q=tongue histology), to find the answer.
Do it. Then if you still have question, give us what answer you came up with and I will tell you my answer.
Edit: You are lucky to be living in Google age, otherwise you would have to go pick up a histology atlas. And that is what you should do anyways.
There are so many things to point out, so I wouldn't. But as you say " i did have very low dna concentrations of vector n insert", I think that could be the reason. YOu should at least have 50-100 ng of linearized, purified plasmid in the ligation reaction. That is crucial. Then comes the 1:3-1:5 molecular ratio of vector:insert. So in your case you need 100 ng vector and 25 ng of insert in your reaction. Or 50+12.5. But don't go below 50 ng for linearized vector.
This is too general a question. There could be one or two of about 74 things that could be going wrong. See here for some PCR array manuals and troubleshooting guides, think critically, get help from a senior colleague to get an objective assessment of your techniques that you may be missing.