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I started a topic yesterday in Bioinfo regarding NCBI blastN, i would like you all to be aware that when blasting against bacterial genome.. you will no longer get the Gene ID/ Genebank associated with the subject sequence that your query is most related to. No more associated gene.. this definitely makes life harder.
It still works for BlastP. For those involved in bacterial genetic and novel gene isolation .. i think our lives have just been rendered slightly difficult. You still could get somewhat info on the gene by turning on the " CDS feature" it will help out abit. In order to download the FASTA sequence, we have to open up the entire genome of the bacteria and key in the region that we would like to get info on instead of clicking on the "linkout" provided last time.
Let me know if any of you are experiencing the similar situation.
you are totally right ...a week ago or so there was a clear link to the gene associated with the hit sequence!
This feature is now missing ...and it was a very important feature to my point of view!!!
I think i should also contact the NCBI support to stress that this feature has to be reactivated!
I did email the NCBI people, after discussing with the developers he said i could turn on "CDS feature" in the formatting option .. but i know it used to be way EASIER!
I wonder why they remove this particularly useful feature. it definitely saves more time.
Let me know what the NCBI people tell you. They usually reply pretty quickly. I was directed to a person called Dr Tao Tao.
IN the end , i have to open the whole genome.. and take out the genome region that i wan according the the number(BOLDED) from the alignment and find the gene ID/CDS from there.
Query 35 TCGGCCCCGGCACGCCCGGCGGCAATCTGCTGCGTCGTTACTGGCAGCCCGTGGCCCTGG 94
||||||| ||||| ||| |||| | ||| |||| | || ||||| || ||||| ||||
Sbjct 2225376 TCGGCCCGGGCACTCCCTGCGGGCAGCTGATGCGCCAGTATTGGCAACCGGTGGCGCTGG 2225317
The NCBI staff insists that i am wrong. but i know it existed because i been doing alot of blast work before this.
IN this picture that i m uploading.. there's NO darkblue box with G to indicate genebank in the links area.
It's all empty. It used to be there when i did it last month.. and i had the " linkout" box ticked.
Make new solution of Ammonium acetate.
After one month this solution goes cuckcoo apparently due to decomposition.
My plasmid prep " resist' digestion after one month from the preparation of my solutions.
b4 that everything was ok.
changing the solution 3 made all the difference.
did you run a gel of your preparation? did it show a band or pattern that you expect for your protein?
you could have a denatured enzyme. you may also be experiencing the presence of a native inhibitor. the enzyme may be too dilute in the extract.
you may have to at least partially purify the enzyme.
According to the literature this sort of dioxygenase system is a multicomponent one. IN any case, from what i have read most group could observe activity when they did the enzyme assay with cell-free extract but using other sort of aromatic compound.
As of now, i do not know whether which enzyme is specific for its reaction. The fact that it could grow on the compound as its sole nitrogen /carbon source should indicate it has the pathway for this degradation. so in my mind, the enzyme(s) should be present.
Currently hoping to develop/modify an existing assay first to determine its activity prior to partial purification.
Thanks for the comment.
i have added glycerol and dtt. i wonder if you have any suggestion as to how to stabilize and prevent its denaturation.
In addition, how to prevent the effect of native inhibitor? that's somewhat new to me
I am assaying for a dioxygenase activity on a aromatic compound , basically a aromatic ring hydroxylating activity.
The cell was grown using the aromatic compound as sole carbon source .
during the mid log phase, i harvested the cell . centrifuged at 4000rpm at 4C for 30 min.
washed with 20mM K/Na Phostphate buffer once
then eventually the cell was resuspended in 50mM Tris-HCL pH 7.5 with 1mM DTT and 10% Glycerol
in order to extract the protein , i used a Vibracell 600W. 10% amplitude 2.5pulse and 2.5 pause. for 10 min. done in ice bath
The lysate is clear. then i proceed to centrifuge at 12,000rpm for 30 min.
The supernatant would be the cell-free extract in my stage.
So from here the reaction goes like this
1ml reaction (containing)
0.2mM Ferrous ammonium sulfate
25mM Tris-HCL pH7.5
0.05 ml Cell Free extract
1mM of aromatic compound
incubate at 30C for 30 min
negative control is the similar reaction but without the substrate.
i compared the abs at 340nm for reduction in NADH.
at the end , i couldn't observe any significant difference.
What could be the problem.
any suggestion would be highly appreciated
1. extracellular enzyme??
2. denatured protein?
3. any other??
it's my first time experience in doing enzyme assay . hope to hear from you guys soon.
to prevent this by acidifying the solution with sulphuric acid to pH 2 or less.
or use ammonium ferrous sulphate. the Ammonium keep the solution acidic to some extent
go with the AT cloning business adn get the individual plasmid as suggested above.
But why should life be simple and without challenges
Thank you all for your comments, finally after a week in + 70 C my buffer seems to be clear!
I also suspect it is EDTA. A week in 70C.. crazy haha.
however if at all time u could keep it simple, please do. Research is a race against time.
i have seen Industrial based ethanol (Cheap stufff) that we would use for flaming or the typical 70% etoh disinfectant being used for DNA-based work.
urg needless to say inconsistent sequencing result, bad digest etc etc.
it will smell different. pure ethanol won't sting the nose as compared to cheap one
This is to avoid having a suboptimal condition for the restriction digest. the salt present in the pcr reaction might affect the final salt concentration required for various restriction enzyme which eventually cause star activity or decrease or even inhibits the reaction.
i have been looking through the tris powder in my lab. particularly those from a particular manufacturer. it appears that the tris powder itself is somewhat yellowish.
an undergrad student make a 50X tae buffer with it and holy smoke, the whole buffer is yellowish...
should that be a concern?
is there any explaination to why the tris powder is yellowish. as far as i am concerned it should be white.
Continue to amplify with your proof reading polymerase. You should purify the product before adding the A overhangs as the pfu will remove them unless you go immediately onto the cloning step.
thanks for reply but how come in long pcr or high fidelity enzyme mixes that are mixture of tag and a proofreading enzyme we can use pcr product directly in t/a cloning? isnt there a risk of a overhang removal?
Well, in long PCR mix, the amount of proof reading polymerase is present in quite small amount. even if it removes it , the dominant Taq will add it back and there might be those without tail but perhaps at very minute amount.
However in your case, i think you might be able to eliminate gel extraction if i m analyzing ur information right. i did make an assumption that the distance between NotI and SaiI cutsite is quite small <50bp perhaps.
Using this assumption here..
What i would do.
AFter u PCR out the gene using primer which will give another more bases before the SaiI site
1. digest vector/ pcr fragment separately
2. NotI and SaiI could be run in the same buffer, both are heat inactivatable, that's good.
3. after digestion, heat inactivate, ethanol precipitation
the idea I came through is that amm.acet is anionic, and so it pulls apart the DNA strands (by disturbing hydrogen bonds between bases). this when combined with low temperature and propanol/ethnaol (non-polar solvents) forces the DNA to precipitate.
with regard to potassium acetate in plasmid extraction, its said that it makes the cell-lysis-solution a little less alkaline (how??) so that this pH is sufficient for the small plasmid DNA to reanneal and not the big genomic DNA.
and finally amm.acet for protein extraction, its said that it decreases the dielectric constant of water, thereby disturbing the hydration layer of proteins, forcing them to precipitate (with addition of isopropanol). I'm not quite sure whats the relation between dielectric constant and hydration layer
potassium acetate pH5.5 usually. it neutralize the solution and renature the nucleic acid which was previously denatured by NaOH. however, genomic DNA will have trouble reannealing properly as they are huge.. eventually they precipitate out of the solution. while plasmid DNA is small and will anneal well and remain in the solution.
combine that with omni taq and u might just get what you need.
Thanks for your help! it worked quiet well!!
Do you know if the final plasmid is clean enough for sequencing and transformation?
if not what should i do to obtain clean quality?
For transformation, definitely . sequencing i would assume it's alright. washing with 70% ethanol usually will provide sufficiently clean DNA. way better than conventional column purification.