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Member since: Jul 12, 2009
From: Georgia, United States
Status: Protein Chemistry & Glycobiology Moderator
My points: 1083    what's this
Name: [Privacy]

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Sep 19, 2015    01:28 AM

Replied to topic in Protein Detection (Western blots, gels, IP) forum   Bubbles in Western Blot Gel

saddav, The bubble is between the gel holders and the gel, correct? Have seen that before, have no explanation, but I don't think those bubbles will affect the final WB outcome. In my hands, I proceed to the transferring and blotting steps and still abe to obtain proper bands. Cheers.

Jul 17, 2015    04:47 PM

Replied to topic in Protein Detection (Western blots, gels, IP) forum   Western Blot using Odyssey Troubleshooting

-- Your Primary has an isotype of IgG2b, your secondary only recognises antibodies with IgG1 isotype. You need to find either anti-mouse IgG2b or anti-mouseIgG (whole IgG regardless of their isotype)-- This will definitely solve your problem. Good luck

Jun 26, 2015    06:14 PM

Replied to topic in Cloning (Recombinant Gene Expression) forum   curved gel band from mini prep

I kind of think 2 ul of miniprep is a tons of DNA.... Can you try to make a 1/10 dilution and load like 1 or 2ul and see whether your gel resolution improves?

Jun 25, 2015    01:07 AM

Replied to topic in Cloning (Recombinant Gene Expression) forum   Any suggestions on why tag is not working in western blot?

Hi tanmiyitank, I suspect your problem rises from invalid western condition- You can try to perform a serial dilution of your primary antibody ranging from 1/5000 to 1/20000 dilution to first optimize the best condition of the anti-flag antibody used in your western. The other thing you might wan ...

Jun 22, 2015    07:25 PM

Replied to topic in Cloning (Recombinant Gene Expression) forum   curved gel band from mini prep

Is it possible you overloaded those lanes? Do you have any idea what is the concentration of your DNA?

May 4, 2015    04:26 PM

Replied to topic in Pharmacology and Drug Discovery forum   isolating dna from spotted on filter paper

Hi Mbiotech, Add ddH20 or TE buffer enough to soak the filter paper wet, incubate at RT (I like to leave it for several hours to overnight, but I think it is over-kill), vortex, spin down. Your DNA will present in the solution and you can use it for transformation to amplify the plasmid. Good ...

Apr 30, 2015    06:06 PM

Replied to topic in Biochemistry forum   Biochemistry

Hi Jadmeteofan, I recommend you read up the following link to help with your problem set from your biochemistry course. Good luck.

Mar 24, 2015    12:48 AM

Replied to topic in Biochemistry forum   Determining Enzyme Kinetics

Tara-linth, To plot Michealis Menten, the x-axis is substrate concentration and y-axis is velocity, Hence, you need to calculate your enzyme velocity (rate at which product is formed). If you have a standard curve where you know the absorbance of your product, you should be able to back calcu ...

Mar 18, 2015    04:32 PM

Replied to topic in DNA (PCR, Real Time PCR) forum   Primers

one additional side note to what creativeproteomics has touched on- I preferred to prepare the working solution of primers with ddH2O. Since the DNA polymerase reaction is Mg-dependent, by doing the 1/10 dilution with ddH2O can ensure the amount of EDTA in the final PCR reaction become insignific ...

Mar 4, 2015    04:46 PM

Replied to topic in Diagnostics (Molecular, Pathological) forum   DNA dilution

Hi jem, Since you start with 1000nmol per 1 ml (or 1000 nmol per 1000 ul), the concentration = 1 nmol per ul. Hence, by taking 5 ul from your stock, it will contain 5 nmole of your oligo.

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