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Pangea's Content

There have been 97 items by Pangea (Search limited from 12-April 20)



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#147484 How do you improve your bio skills?

Posted by Pangea on 04 January 2013 - 03:52 PM in General Lab Techniques

Well learning by
doing. Do a lot of mistakes and you will got it. Read and Repeat



#147948 Stem Cell Donation

Posted by Pangea on 13 January 2013 - 07:40 AM in Stem Cell

Where can I type me and donate stem cells?



#147957 Stem Cell Donation

Posted by Pangea on 13 January 2013 - 09:28 AM in Stem Cell

Germany



#147963 Stem Cell Donation

Posted by Pangea on 13 January 2013 - 09:40 AM in Stem Cell

Cheers on both.



#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.



#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!



#147431 Can I make an SDS stock in media and store?

Posted by Pangea on 04 January 2013 - 05:44 AM in Tissue and Cell Culture

Sds can precipitate in low temperature. Heat or vortex.



#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?



#147942 pEGFP C-1/N-1 Cloning

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

Blunt or sticky Ends?



#147947 Purifying a "protein"

Posted by Pangea on 13 January 2013 - 07:12 AM in Biochemistry

Run a SDS-PAGE and do Silver-Staining. Measurement on 280 nm for Purity.



#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?



#147443 Problem with cloning - what is wrong?

Posted by Pangea on 04 January 2013 - 08:43 AM in Molecular Cloning

Well , check your primer again but it is always better to have two different RE . You do not have a direction problem or multimerization of insert. Furthermore, your insert and vector is really big in size, so check again different ligation condition. The big colonies mean that your CIAP reaction was not completly successful.



#147430 cloning: band at different site than desired after RE digestion

Posted by Pangea on 04 January 2013 - 05:41 AM in Molecular Cloning

Q5: It seems that NdeI is having some difficulties cutting my DNA. Is there a reason for that?A5: NdeI activity is sensitive to contaminants present in DNA isolated with standard miniprep protocols. Further purification of the DNA by a column or dialyzing the contaminant from the DNA may help increase the activity of the enzyme on the isolated DNA substrate.Q6: NdeI seems to be digesting my DNA correctly, but when I try to ligate it, I obtain no colonies. Is there a potential explanation for what I am observing?A6: NdeI is a very robust enzyme. If the substrate DNA is digested for extended periods of time, we have found evidence that the enzyme will remove some additional nucleotides. Digestion of DNA with NdeI over 4 hrs is not recommended.

Maybe your gene is multiple cloned into the vector.And using 2 or 3 colonies is pretty few. And your primer might be not specific enought.



#147561 cloning: band at different site than desired after RE digestion

Posted by Pangea on 06 January 2013 - 01:07 PM in Molecular Cloning

You have right you have single cut RE. Sorry about confusing.



#147497 cloning: band at different site than desired after RE digestion

Posted by Pangea on 05 January 2013 - 01:18 AM in Molecular Cloning

It might be that your digestion of your Insert is not working out you have blunt ends. So it will just ligated several inserts. But i am not sure. And i hope you are not using BL21 for plasmid replication. Not recA mutation. But BLR have it or other K 12 strains. And are sure that you are not cutting into your plasmid?



#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.



#147532 Insulin production

Posted by Pangea on 05 January 2013 - 12:59 PM in Protein Expression and Purification

What would be the best way to express and purify active insulin?



#147751 Insulin production

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

Cool source thaks for the hint.



#147513 High Yield Protein Expression 10g/L

Posted by Pangea on 05 January 2013 - 06:47 AM in Protein Expression and Purification

It will be a pET vectorsystem for E coli. One can play with media and ZnCl2, Glucose and VPA. But is that enought for very high yield production.?



#147958 High Yield Protein Expression 10g/L

Posted by Pangea on 13 January 2013 - 09:30 AM in Protein Expression and Purification

So you mean replacement by centrifuging the cells and renewing the media. Can you give me more details like an protocol?



#147388 High Yield Protein Expression 10g/L

Posted by Pangea on 03 January 2013 - 10:06 AM in Protein Expression and Purification

Hi,

how can İ best get in E. coli or Mammalian cells an protein expression yield of more than 1g/l?

Any kind modifications?

Cheers



#147593 my competent cell grow on any agar plate

Posted by Pangea on 07 January 2013 - 07:32 AM in Microbiology

Under which condition are you growing the stock solution cells before transforming with plasmid to have enought cells? Or are you using a stock aliqout?



#147429 my competent cell grow on any agar plate

Posted by Pangea on 04 January 2013 - 05:27 AM in Microbiology

Are you sure that your agar plates are alright.? Do you have positive and negative controls.? And check your protocol doing the competent cells.



#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?



#147961 Alternative to pET Vectorsystem

Posted by Pangea on 13 January 2013 - 09:35 AM in Molecular Cloning

Any suggestion about other vectorsystem than pET?




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