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kant0008's Content

There have been 86 items by kant0008 (Search limited from 06-August 19)

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#7991 RNA Isolation

Posted by kant0008 on 18 October 2004 - 11:04 PM in Molecular Biology

Why not give it a go with a couple of samples? Also depends on what exactly you are planning to do with it, some techniques are more sensitive than others to contaminants and degradation.

#7992 plasmid digest

Posted by kant0008 on 18 October 2004 - 11:07 PM in Molecular Biology

Are you sure that your plasmid is what yopu think it is?
How many sites for EcoRV is in it?

#9253 semi-dry transfer

Posted by kant0008 on 07 December 2004 - 08:58 PM in Protein and Proteomics

if you also swapped over the anode/cathode wires (plugged into the wrong side, or up in the powerpoint) your blot would work as normal. That's the only thing I can think of at the moment.

#12782 MMP assay

Posted by kant0008 on 22 March 2005 - 09:40 PM in Cell Biology

You could make cell extracts- cytoplasmic for eg, without adding protease inhibitors, as long as you work quickly and in cold conditions it shoul dbe ok.

#7665 Digestion time and enzyme

Posted by kant0008 on 05 October 2004 - 09:24 PM in Molecular Biology

as for time- how long is too long? Some enzymes have "star" activity, they start cutting non-specifically if at too high concentration or if left too long, I'm not sure if Bam HI has it though. I don't think there are any specific requirements for cutting PCR products.
To get rid of your enzyme you can ethanol precipitate your mixture.

#7473 question about stable transfection

Posted by kant0008 on 26 September 2004 - 04:23 PM in Cell Biology

it depends on your cell growth also, but G418 is stable for about 4 days, so if your cells don't become overconfluent or start looking sick and like they need a "feed" once in 4 days should be ok

#6425 Bands are like a "w"

Posted by kant0008 on 08 August 2004 - 05:09 PM in Molecular Biology

normally strange band shape is to do with the gel, for example agarose not dissolving properly before you por so there are clumps, or setting slightly before you pour- again causing small clumps that would cause the sample to run unevenly. Try decreasing the agarose conc and make sure it all dissolves properly. It could theoretically be a problem with your power supply, but then you'd get a strange pattern all over your gel, not the same for all bands.

#6315 his and myc tags

Posted by kant0008 on 02 August 2004 - 06:20 PM in Protein and Proteomics

A his-tag is a technique used in biochemistry to purify proteins. The protein to purify is expressed as a fusion protein, carrrying a series of histidine residues at either the amino or the carboxyl end. The name-giving histidine residues will stick to a nickel surface, and can be eluted from there via imidazol. His-tagged proteins can also be identified on a Western blot via -his-antibodies.
Myc tags are similar.

#7028 Western Blots

Posted by kant0008 on 08 September 2004 - 08:42 PM in Protein and Proteomics

Incubation with milk in theory should give you a cleaner western with less background noise

#6383 vector with no sp1 in promoter

Posted by kant0008 on 05 August 2004 - 05:45 PM in Molecular Biology

I'm trying to find a plasmid vector with some sort of a detectable product that has no sp1 sites in its promoter, as I'm trying to monitor the effect of sp1 on my gene of interest and want to find a plasmid I can use as a transfection efficiency control.
Thanks for any suggestions!

#7127 ELISA normalisation

Posted by kant0008 on 13 September 2004 - 11:12 PM in Protein and Proteomics

Hey all,
just wondering, when you do ELISA do you dilute your samples to a uniform total protein concentration and then do the assay, or is it ok to use eg. a constant volume of sample and then work out the concentration of protein of interest later, based on a Bradford?

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