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Pangea's Content

There have been 97 items by Pangea (Search limited from 10-April 20)



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#147942 pEGFP C-1/N-1 Cloning

Posted by Pangea on 13 January 2013 - 06:31 AM in Molecular Cloning

Blunt or sticky Ends?



#147941 TAP Water

Posted by Pangea on 13 January 2013 - 06:23 AM in General Lab Techniques

It will be serious check. Cool manual.



#147920 No activity of expressed protein obtained from pET32a cloned gene

Posted by Pangea on 12 January 2013 - 01:49 PM in Protein Expression and Purification

Why do you have TRX tag if you are purifying with His Tag? Is it to cleave the His-Tag or what? Are you using ELISA for hte activity assay?



#147919 pEGFP C-1/N-1 Cloning

Posted by Pangea on 12 January 2013 - 01:43 PM in Molecular Cloning

Are you plating all bacterial cells or just taking 100 ul of your Sample?



#147918 TAP Water

Posted by Pangea on 12 January 2013 - 01:36 PM in General Lab Techniques

How can I analyze tap water for microbes?



#147911 Albumin Removal From BSA

Posted by Pangea on 12 January 2013 - 12:22 PM in Protein Expression and Purification

You can use Affinity Chromatography or Ion Exchange Chromatography (Isoelectric Point). If you have less amount maybe with sepharose beads.



#147908 Wet transfer of proteins between 100-200kDa

Posted by Pangea on 12 January 2013 - 12:01 PM in SDS-PAGE and Western Blotting

I think 120V for 1.30 - 2.00 h. Maybe should put PVDF in methanol first before transferring. Always check your protein ladder/marker.



#147907 Extraction

Posted by Pangea on 12 January 2013 - 11:58 AM in Botany and Plant Biology

I am just intersted and ask you which part and what you want to extract?



#147906 How to raise polyclonal antibodies against whole bacteria live &/ or inactiv

Posted by Pangea on 12 January 2013 - 11:50 AM in Immunology

In which Organism will you perform your experiment? Hamster, Mouse, Rabbit etc.? Can be tricky with polyconal, am I wrong?



#147905 Software for designing cell pathways

Posted by Pangea on 12 January 2013 - 11:41 AM in Bioinformatics and Biostatistics

I think people are doing for there own signalling pathway by Illustrator. But that takes a liitle bit time. Maybe this website will help you:



http://string-db.org/

http://www.cellsigna...hway/index.html



#147904 How to know which expirements to do?

Posted by Pangea on 12 January 2013 - 11:32 AM in Research Idea, Design and Collaboration

Well, generally they are expecting that you have an overview about that what you will doing in your experiments. Understanding the techniques which you will use to get you to your target/aim. Normally, they should give you an main project which will be splitted or added into small project during your Phd Journay.

Its not easy with your description to make an clear research statment. You know that breat cancer is well studied field in oncology. So you must specify little bit more.

Field in which you will be involved are:

1. Molecular Biology - Recombinant DNA Technology, Cloning, PCR, qPCR, DNA Microarray, etc.
2. Biochemistry - Recombinant Protein Technology, SDS-PAGE, Wester-Blot, IP, Pulldown etc.
3. Cell Biology - Primary Cell, Immortalized Cell Line, Transient/Stable Transfection, siRNA and/or shRNA, MTT Test, Apoptosis Test, Proliferation Assay etc.
4. Screening - small molecules Library, Inhibitors etc.

If i could help you in this way.



#147903 Open-Source Mathematics in Research

Posted by Pangea on 12 January 2013 - 10:45 AM in General Lab Techniques

Wow thats the point. Very useful. Cheers Mate.



#147896 Membrane Protein SDS PAGE and WB

Posted by Pangea on 12 January 2013 - 07:39 AM in SDS-PAGE and Western Blotting

Did you do Coomassie Staining or Ponceau Staining whether its there or not? check this, Which membrane aere using for the transfer? Its maybe just a technically problem.



#147895 Open-Source Mathematics in Research

Posted by Pangea on 12 January 2013 - 07:35 AM in General Lab Techniques

Do someone has a good source for general mathematics in the Lab?



#147859 What is Ks?

Posted by Pangea on 11 January 2013 - 07:00 AM in General Lab Techniques

Best answer give you google, isnt?



#147858 what is the contamination in buffers

Posted by Pangea on 11 January 2013 - 06:59 AM in General Lab Techniques

Are the buffers bought from a company and is there SDS inside? One PhD in our Lab changed the whole Kit because she was not able to read. How stuipid, isnt? Generally, most buffer are store in RT. If there is contamination then it is quite a old solution. Try to for cell culture always aotuclaving the solution and the others in the Lab as you know wiht ddH2O and store tightly closed. Make new once if frequently used. To my knowledge like TAE or TBE or Running Buffer you can make stock solutions so there is no chance to growth for contaminants. And its not specific for TRIS its just a matter of handling your solution. If i was able to help you.



#147825 Large-Scale Purification of Non-Secreted Protein in E.Coli or CHO Cells

Posted by Pangea on 10 January 2013 - 11:17 AM in Protein Expression and Purification

Thanks guy that gives me a better understanding how things are handled. Keep in touch.



#147762 Can I make an SDS stock in media and store?

Posted by Pangea on 09 January 2013 - 08:01 AM in Tissue and Cell Culture

Your welcome!



#147756 Inclusion body formation of the recombinant protein expressed in pET28a in E.col

Posted by Pangea on 09 January 2013 - 06:22 AM in Protein Expression and Purification

Maybe your T7 Promotor is to strong. The more is less and the less is more sometimes. And you can use a protein tag to make your ptotein soluble. Check for this pET-Manual. Use Glucose if your gene is toxic as an addition option.



#147752 Large-Scale Purification of Non-Secreted Protein in E.Coli or CHO Cells

Posted by Pangea on 09 January 2013 - 05:50 AM in Protein Expression and Purification

Yes i know Sambrook and Maniatis. But you missed the sign behind 1000 . "Liters". But RIPA is quite a strong Lysis Buffer. However you say that there is no other technique to break the cells in high amount conc. than Lysis Buffer?



#147751 Insulin production

Posted by Pangea on 09 January 2013 - 05:46 AM in Protein Expression and Purification

Cool source thaks for the hint.



#147750 Dam Dcm Negative Strain

Posted by Pangea on 09 January 2013 - 05:41 AM in Molecular Biology

Can you give some example like JM110 or others? Which strain is most frequently used in the Labs?



#147701 Dam Dcm Negative Strain

Posted by Pangea on 08 January 2013 - 01:14 PM in Molecular Biology

I red that methylated DNA has less tranformation efficiency than unmethylated. How can i get my plamid unmethylated?



#147665 Replication of the Plasmid DNA

Posted by Pangea on 08 January 2013 - 07:24 AM in Genetics and Genomics

:)



#147660 Restriction Enzymes

Posted by Pangea on 08 January 2013 - 01:56 AM in Molecular Biology

Ok , lets say i want gene sequence of an RE from IMAGE consortium. And produce my own RE in E.coli. I am no considering the cost and sso on.. . Question is if its practcally possible in the LAB.




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