Find content
Male
First you have to answer a question
Do you plan to work with cDNA (without introns) or a full size gene?
Human gene tend to have splice site variant and contain regulatory elements within their introns. A cDNA will not be able to capture these facets.
On the other hand full size eukaryote tend to be very big and not easy to manipulate. cDNA tend to be much smaller.
Depending on your answer, you may use one these as a template
a plasmid from a cDNA libraries
a plasmid from a BAC libraries
raw human DNA.
So where can you get a template?
You may either purchase the template DNA or obtain it from a lab working in the field. Using pubmed find out who has publish work on your protein of interest and email them. Invitrogen and Open Biosystems are examples of places where you may purchase a BAC libraries. A google search will turn up more places which sells cDNA or BAC libraries.
Working with raw human DNA is not easy. Stick with libraries.
#114068 Newbie- choose DNA template
Posted
on 02 July 2011 - 05:02 AM
#130551 Cosmetic recall
Posted
on 06 March 2012 - 05:30 PM
The etiology of asthma is not that clearly assocated with antigen exposure and it is a red herring as supporting the hygiene hypothesis. Sterility is an absolute so the term is inapropriately applied - but ';d sure appreciate theday care citation.so we can discuss it. Clearly, most of the work supporting the concept is designed to supportit rather than test it - about what one would expect from MDs..
As I said, there are no data (more correctly, I' not aware of such data and I have searched for it) that living in environments such as a farm exposes one to substantially greater immunoligic stimulus - and the differecne would have to be exponential both in exposure and effect to be clinicaly significant..
As I said, there are no data (more correctly, I' not aware of such data and I have searched for it) that living in environments such as a farm exposes one to substantially greater immunoligic stimulus - and the differecne would have to be exponential both in exposure and effect to be clinicaly significant..
#107248 Self-circularization-How to prevent other unwanted ligations
Posted
on 19 April 2011 - 10:13 AM
You can calculate this. DNA is 0.34 nm/bp long, so your 300 bp fragment will be 100 nm long. You want the probability of finding its own end to be higher than the probability of finding some other end. The local concentration of your end is 1 molecule/sphere of radius 100 nm. Volume is 4/3 pi r^3 or about 4e6 nm^3. There are (10^8)^3 nm^3/liter or 10^24 nm^3/liter. So the local concentration of ends is about 1e24/4e6 molecules/liter = .25e18 molecules/liter = .25e18 molecules/6e23 molecules/mole = .04e-5 molar = 4e-7 molar = 400 nM.
So, your molecular concentration should be significantly below 400 nM. For a 300 bp fragment, the MW is 660*300 = 1e5 Dalton. 400 nM of your molecule will be 400e-4 g/l = 400e-10 g/ul = 40 ng/ul.
This agrees with my intuition that you should be below around 10 ng/ul, perhaps at 5 ng/ul of reaction.
So, your molecular concentration should be significantly below 400 nM. For a 300 bp fragment, the MW is 660*300 = 1e5 Dalton. 400 nM of your molecule will be 400e-4 g/l = 400e-10 g/ul = 40 ng/ul.
This agrees with my intuition that you should be below around 10 ng/ul, perhaps at 5 ng/ul of reaction.
#106723 Salary
Posted
on 13 April 2011 - 09:07 PM
I cant really do anything.... its just been about 6 months into my job (its also my first one).
So I can do nothing about it. But yes, I would like to know and learn how to negotiate salaries for my future. I cant accept (or afford) low wages throughout my life.
So I can do nothing about it. But yes, I would like to know and learn how to negotiate salaries for my future. I cant accept (or afford) low wages throughout my life.
#106410 gelatin staining with coomassie: how do I make it work?
Posted
on 11 April 2011 - 02:46 AM
Hi everybody,
I have been trying to set up gelatin zymography in my lab and ran into a strange problem. When I previously stained gels with a really old and dark Commassie solution somebody once gave me, the gelatin inthe SDS polyacrylamide gel stained nicely, giving a dark blue background over which I could see MMP digestion bands. When I ran out of this solution, I bought Bio-Rad Biosafe Coomassie, just to find out that no matter how long I incubate the gel with it, it will stain the protein bands but it never stains the background, making it impossible to discern any digestion bands...
Any suggestion as to why this happens? Why would gelatin not be stained by this type of Coomassie?! (I use 300 bloom gelatin at a final concentration of 1 mg/ml)
I am willing to buy a different Coomassie, but which one would be best to overcome this problem?
Thanks!
I have been trying to set up gelatin zymography in my lab and ran into a strange problem. When I previously stained gels with a really old and dark Commassie solution somebody once gave me, the gelatin inthe SDS polyacrylamide gel stained nicely, giving a dark blue background over which I could see MMP digestion bands. When I ran out of this solution, I bought Bio-Rad Biosafe Coomassie, just to find out that no matter how long I incubate the gel with it, it will stain the protein bands but it never stains the background, making it impossible to discern any digestion bands...
Any suggestion as to why this happens? Why would gelatin not be stained by this type of Coomassie?! (I use 300 bloom gelatin at a final concentration of 1 mg/ml)
I am willing to buy a different Coomassie, but which one would be best to overcome this problem?
Thanks!
#104743 How do you use tips???
Posted
on 25 March 2011 - 01:48 AM
Ok, something I did while waiting for my PCR....
For
Pito
pDNA
Casandra,
Hobglobin,
mdfenko,
phage434,
...phage was too hard to be "tips engineered"...
Homebrew,
And specially for Ameya
Done by,
For
Vote + anyone?
For
Pito
pDNA
Casandra,
Hobglobin,
mdfenko,
phage434,
...phage was too hard to be "tips engineered"...Homebrew,
And specially for Ameya
Done by,
For
Vote + anyone?
#104109 Help identifying some organisms
Posted
on 18 March 2011 - 03:36 PM
joe:
I am a mycologist. Suggest you isolate these fungi - it's not so easy to identify without colony morphology. That said - I think I see some Fusaria as well as what might be a dematiaceous fungus with alot of Gram negative bacteria.
I am a mycologist. Suggest you isolate these fungi - it's not so easy to identify without colony morphology. That said - I think I see some Fusaria as well as what might be a dematiaceous fungus with alot of Gram negative bacteria.
#104110 Gram Positive and Gram Negative
Posted
on 18 March 2011 - 03:39 PM
Hardly gt - you misled re. Mannitol salt agar. Staph will grow but many Gram positive bacteria will be inhibited by the high salt.
#103640 please correct
Posted
on 15 March 2011 - 12:38 AM
claritylight, on 14 March 2011 - 02:35 PM, said:
Since we fixed my number mistake now, the given is 1ug/ul of stock DNA and I have 10ul so I have 10ug DNA amount right? I was just given the numbers of 10ug and 10ul
Just tell me if the below calculations are correct and I will figure out what I want on my own and we can all move on:
If I put 10ul of the 1ug/ul DNA into a tube, add 190ul water, this = 200ul total volume, and the DNA in this tube
now has concentration of (10ul/200ul)*1000ng/ul = 50ng/ul right?
What I want to know is how much amount of DNA is in the tube. I did 50ng/ul*200ul = 10000ng = 10ug, so there's 10ug of DNA in the tube. Is that right?
Just tell me if the below calculations are correct and I will figure out what I want on my own and we can all move on:
If I put 10ul of the 1ug/ul DNA into a tube, add 190ul water, this = 200ul total volume, and the DNA in this tube
now has concentration of (10ul/200ul)*1000ng/ul = 50ng/ul right?
What I want to know is how much amount of DNA is in the tube. I did 50ng/ul*200ul = 10000ng = 10ug, so there's 10ug of DNA in the tube. Is that right?
Ok, now I get it, see: telling you have 10µl of a 1µg/µl stock is not the same as just saying: 10µl of 10ng...
10µl of 1µg/µl in a volume of 190µl means you have in total 10µg in 200µl thus 10µg/200µl = 1/20µl = 1000ng/20µl = 100ng/2µl = 50ng/µl.
this is what homebrew allready did.
So yes, if you have 50ng/µl and you have 200µl , yeah, you have 50*200 ng DNA= 10000ng = 10µg...
But I dont understand why you do this second calculation!
You started by saying you would add 10µl of a 1µg/µl solution in that tube... So you knew from the start it was 10µg ..
(10µl*1ng/µl = 10ng .. and no matter how much water you add, even its 19999999liter, it will still stay 10ng of DNA in the end... Its the concentration that changes, not the amount of DNA itself)
You calculate B , using A to calculate A in the end ...
But still: I have the feeling you dont understand the formula or that you dont know what you are doing.
#101329 DNA, Gel
Posted
on 20 February 2011 - 11:51 AM
sxh999, on 20 February 2011 - 10:37 AM, said:
Thank you all for your reply.
I meant I have problem to attach the image of my Gel in this website.
I will try to poster the image here, so you can have a look and help me to analyze. thank you very much.
I meant I have problem to attach the image of my Gel in this website.
I will try to poster the image here, so you can have a look and help me to analyze. thank you very much.
#101258 DNA, Gel
Posted
on 19 February 2011 - 10:26 AM
I was not permitted to upload my gel image.... 
#101259 DNA, Gel
Posted
on 19 February 2011 - 10:27 AM
sxh999, on 19 February 2011 - 10:26 AM, said:
I was not permitted to upload my gel image....
What was the error if you had one?
Maybe the file was too big?
With a picture its always easier to get help.
#101268 DNA, Gel
Posted
on 19 February 2011 - 11:56 AM
pito, on 19 February 2011 - 10:27 AM, said:
sxh999, on 19 February 2011 - 10:26 AM, said:
I was not permitted to upload my gel image....
What was the error if you had one?
Maybe the file was too big?
With a picture its always easier to get help.
Maybe it's because they're a new poster. Newcomers often have such restrictions on forums until their post-count hits a certain number.
#101328 DNA, Gel
Posted
on 20 February 2011 - 10:37 AM
Thank you all for your reply.
I meant I have problem to attach the image of my Gel in this website.
I will try to poster the image here, so you can have a look and help me to analyze. thank you very much.
I meant I have problem to attach the image of my Gel in this website.
I will try to poster the image here, so you can have a look and help me to analyze. thank you very much.
#101495 copy number of gene in genome
Posted
on 22 February 2011 - 03:00 AM
The number of a particular gene present per genome does not change due to expression level -- expression level changes influence the number of RNA transcripts produced from the gene and, ultimately, how much of that gene's product (protein) is produced, but not the number of copies of the gene itself.
So, while expression level does not change the gene's copy number, the copy number of a gene in a genome can influence the expression level -- if you have two copies of a particular gene in a genome, the expression level of that gene would be double relative to a strain which has only one copy of the gene (all other things being equal).
So, while expression level does not change the gene's copy number, the copy number of a gene in a genome can influence the expression level -- if you have two copies of a particular gene in a genome, the expression level of that gene would be double relative to a strain which has only one copy of the gene (all other things being equal).
