RE: Does Taq accespt ATP as substrate?

Taq does not need ATP for its reaction unlike ligases. Moreover, I doubt whether it has an ATP binding motif at all. Jay -Original Message- From: methods-boun...@oat.bio.indiana.edu [mailto:methods-boun...@oat.bio.indiana.edu] On Behalf Of DK Sent: Friday, February 15, 2013 7:53 PM T

RE: RNA shipment

ments didn't come straight from the companies responsible for the machines, or paranoiac state agencies in the US. Also if routine reviews didn't keep discovering machines outputting orders of magnitude more radiation than intended. On 19/09/12 14:21, Jayakumar, R wrote: > The ai

RE: RNA shipment

The airport scanners are usually backscatter X-ray machines that have much less ionizing radiation than the regular transmission ones used in hospitals. Recent research into these airport scanners show that you have to be exposed atleast 10 million times a year before you have a chance of getti

RE: Overgrown (3day) LB plates ok to use?

If they were antibiotic resistance plates, they are probably useless and full of satellite colonies. Jay -Original Message- From: methods-boun...@oat.bio.indiana.edu [mailto:methods-boun...@oat.bio.indiana.edu] On Behalf Of David Liu Sent: Monday, August 06, 2012 7:39 PM To: meth...@magp

RE: Hello...problem of detecting proteins with CBB staining

Perhaps you should put that question to the author. I have never felt the need to heat up a gel to stain it with CBB. Silver staining is still the most sensitive method to detect proteins, and if it si working only intermittently, perhaps you should look at your handling procedures. And

RE: regarding volume to be maintained in 12ml ultracentrifuge tubes during ultracentrifugation

Depends. The ones that seal on top can take full volume. The ones that do not and is open should not exceed 75% of the stated volume. Jay -Original Message- From: methods-boun...@oat.bio.indiana.edu [mailto:methods-boun...@oat.bio.indiana.edu] On Behalf Of divya teja Sent: Monday, Ma

RE: DNA sequence alignment

Try using Philip software for the phylogeny. I have used this along with ClustatW for several years now and always gives expected results relative to the alignment. It just might be the algorithm used by mega5 weights the pairing differently from what we visualize from an alignment. Jay

RE: Regarding primary antibody binding in western blot

I have also observed people probing multiple PVDF blots separated by a nylon mesh to prevent the blots from sticking to each other, in a box that is just larger than the blot itself. I thought that was a neat idea. You can try that way too. Jay -Original Message- From: methods-boun

RE: Inoue 1990 original paper for SEM transformation

You can also read the Current protocols in Molecular Biology. It is in there. Jay -Original Message- From: methods-boun...@oat.bio.indiana.edu [mailto:methods-boun...@oat.bio.indiana.edu] On Behalf Of DK Sent: Tuesday, May 01, 2012 4:36 PM To: meth...@magpie.bio.indiana.edu Subject: Re:

RE: 10X PBS

My pleasure Pow :-) Jay -Original Message- From: methods-boun...@oat.bio.indiana.edu [mailto:methods-boun...@oat.bio.indiana.edu] On Behalf Of Pow Joshi Sent: Monday, January 09, 2012 3:18 PM To: methods Subject: Re: 10X PBS I like this discussion. A lot of info about old forgotten phys

RE: 10X PBS

Very true, but the change in pH is really not that much within the pKa range and is deionised water has a lower impact on their activities than tap water or distilled water has. I have not tested this and hence I hope I am right :-) Please correct me if I am not. Jay -Original Message---

RE: pH irregularity in buffers

Philip, Indeed, temperature affects pH a lot, a concept quite forgotten by many researchers. pH should be estimated at the appropriate temperature especially when preparing certain buffers like protein lysis buffers which require a specific pH at much cooler temperatures for cell lysis. Adj

RE: 10X PBS

buffers here... DK > > >On Wed, Jan 4, 2012 at 3:38 PM, DK wrote: > >> In article , "Jayakumar, >> R" wrote: >> >Ideally the pH should not change much between 10X and 1X if deionised >> water was >> > used for dilution. Presnce of NaCl h

RE: 10X PBS

l chemistry, just general. If you take 10X PBS and dilute it with deionized water, it will certainly change the pH. For a 10-fold dilution, it should change the pH by approximately 1 (log scale). On Wed, Jan 4, 2012 at 3:38 PM, DK wrote: > In article , "Jayakumar, > R" w

RE: 10X PBS

Ideally the pH should not change much between 10X and 1X if deionised water was used for dilution. Presnce of NaCl has nothing to do with pH changes since it contributes neither hydroxyl nor hydrogen ions (unless otherwise it is contaminated with something else), the balance of which determines

RE: 10X PBS

You might be referring to the use of PBS in animal systems like mice or rat. When they mean PBS i.p. (was it written this way?), it means administration of PBS into animal was via intraperitoneal injection or i.p (in the abdmomen). Unless otherwise told, PBS contains both KCl and NaCl along w

RE: Hello...Native Page is not polymerising

It is not the lack of polymerization that this "myth" is talking about. Many years ago, towards optimising semi-quantitative conditions for westerns, I did a detailed study on all the variables that affect polymerization of polyacrylamide including efect of tris, sds, polyerization temperatures

RE: Hello...Native Page is not polymerising

Sudheer, If you need help, you need to provide information on what you did. You cannot ask people to read up articles for you. Please provide the protocol briefly. If a gel does not polymerise, it is usally something to do with the proton donor used and the catalyst, whcih are usually APS

RE: Sterilised LB media getting some medicine smell, while I was growing E.coli culture

Sudheer, Medicine smell ?? Medicines smell differently. Can you describe the smell better? Did the colour of the media change after you autoclaved it? And how long did you autoclave it? Jay -Original Message- From: methods-boun...@oat.bio.indiana.edu [mailto:methods-boun...@oat

RE: Amplify Software Program and Mac OS X Lion

There are large number of programs to check primers on the web. There were great algorithms around the web long before programs like amplify. I would use Netprimer to check out primers. But also search the site www.expasy.ch for more free online tools. Jay -Original Message- Fr

RE: mammalian stable cell-line

WWw.atcc.org From: methods-boun...@oat.bio.indiana.edu [methods-boun...@oat.bio.indiana.edu] On Behalf Of Pepa Florez Pérez [snipeur...@hotmail.com] Sent: Friday, June 24, 2011 3:58 PM To: methods_magpie Subject: mammalian stable cell-line

RE: Immunochemistry question (probably dumb)

Peter, WHen (or IF) you get the response, please share it with us. Jay From: methods-boun...@oat.bio.indiana.edu [methods-boun...@oat.bio.indiana.edu] On Behalf Of Peter Ellis [pj...@cam.ac.uk] Sent: Tuesday, June 21, 2011 6:53 AM To: meth...@magpie.bio.indiana.

RE: Immunochemistry question (probably dumb)

Secondary antibodies may have also been adsorbed against each other to ensure specificity before being recommended for double staining. As Wo suggested, (Fab)2 fragments missing their Fc fragments but directed against FC of primary may also be used to prevent non-specificity. OR IT MAY JUST BE

RE: Copper-Glycine buffer(Ph-7.4)

Also remember that Copper dichloride do form copper hydroxide (precipitates)with a base such as NaOH as the other gentleman suggested. Jay -Original Message- From: methods-boun...@oat.bio.indiana.edu [mailto:methods-boun...@oat.bio.indiana.edu] On Behalf Of WS Sent: Tuesday, April 26,

RE: Copper-Glycine buffer(Ph-7.4)

Do you really need that high molarity for both salts. That might explain the precipitation. Rarely is copper chloride or glycine (max of 1 M) used at that concentration for any buffers. Please double check your recipe. Perhaps Copper dichloride is at 0.1 to 0.3 M. If you are trying to make

RE: Can anyone please tell about slope function

Thanks Josh, This is what I am trying to say as well. I have been a member of this listserv for well over 13 years now, since I was a student myself. During my graduate days, I have asked several silly questions as well, and there has always been some kind gentleman who took the time to an

RE: Can anyone please tell about slope function

I am not surprised. I work in the US and I also handle a lot of graduate students in the biological sciences as well as in the field of business administration. About 85% of these students have no understanding about linear regression or about slope or even if they know it, they have no underst

RE: Can anyone please tell about slope function

H!! the protein concentrations are on the X-axis and OD on the Yaxis. Always dependent variables on the Y-axis. Jay From: methods-boun...@oat.bio.indiana.edu [methods-boun...@oat.bio.indiana.edu] On Behalf Of WS [novalidaddr...@nurfuerspam.de] Sent

RE: Can anyone please tell about slope function

SLope is a function of how much the dependent variable Y changes for every unit increase in the value of the independent variable X. So the line may be negative or positive (negative if Y and X are inversely correlated). Knowing that, imagine that the slope of the line you are trying to calcul

RE: quantifying PCR bands

Try using ImageJ. It is a standard scientific way of quantifying bands. But if you have access to a laser densitometer, then that is the best way to do it with the Imagequant software (in the case of molecular dynamics model). Otherwise, it is scientifically acceptable to use ImageJ or Scion I

RE: Request

You might be getting RNAse contamination, which is very difficult to eliminate except if you use dedicated workspace for RNA work. You should use clean labcoats, workspace that has been wiped down thoroughly with agents such as RNAseOUT or RNASE-BIOHIT or some other regaent that can remove RNAs

RE: Sealing Tape for Petri Dishes?

Parafilm works best. You have to check out with a microbiologist and he will show you how to use the parafilm to seal plates. You will love it once you know how to do it correctly. The half an inch to 1 inch width cutouts should be held against the edge of the plate with left thumb and then u

RE: Blood is settling

Since you are studying platelet adhesion, do you need red blood cels around. YOu could remove them by sedimenting them out and then use the plasma for your flow studies. jay From: methods-boun...@oat.bio.indiana.edu [methods-boun...@oat.bio.indiana.edu]

RE: Saponin replacement?

Zap them in the microwave :-) Jay -Original Message- From: methods-boun...@oat.bio.indiana.edu [mailto:methods-boun...@oat.bio.indiana.edu] On Behalf Of WS Sent: Tuesday, July 27, 2010 5:12 PM To: meth...@magpie.bio.indiana.edu Subject: Re: Saponin replacement? Dear Joshua, If you jus

RE: Saponin replacement?

Probably you are talking about saporin (saponin is a detergent while saporin is a toxin). You cannot use Tween-20 or Triton X-100 as well, since they are also detergents and will lyse the cells. Best of luck Jay -Original Message- From: methods-boun...@oat.bio.indiana.edu [mailto:m

RE: plasmid from non viable glycerol stock

What percentage glycerol did you use nad was this frozen at -80C? I use 40% glycerol to freeze and it lasts for years and years with good viability. Moreover, it is important that you never thaw the glycerol stock inbetween uses. That reduces viability too. I doubt whether you can retrieve th

RE: anti-phospho S, T & Y antibodies

We routinely do phosphotyrosine blots (human proteins) with anti-phosphotyrosine antibody clone 4G10 (many companies sell this). This is the best and most cited antibody for phosphotyrosine and the most specific too. It is of course not specific to any particular protein. Jay -Origi

RE: IHC with Nalm-6

Welcome to the world of IHC. They only like nice in publications. You got to keep trying different antibodies before you can get one that works. Every company claims that their antibodies work, but take that with a pinch of salt. Usually, they don't. Also check out literature and contact peop

RE: Cloning trick

You mean you want to linearise the construct by cutting it with a RE outside the expression cassette before transformation??? Is that what you mean. I don't see the purpose of cutting a vector after ligating vector and fragment to clone a PCR fragment efficiently. It does not make much sense.