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Pangea's Content

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



#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?



#147942 pEGFP C-1/N-1 Cloning

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

Blunt or sticky Ends?



#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.



#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.



#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?



#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.



#147974 Difficult Ligation - Details Inside

Posted by Pangea on 13 January 2013 - 12:41 PM in Molecular Cloning

I already changes my mistake. Sorry.



#147980 Difficult Ligation - Details Inside

Posted by Pangea on 13 January 2013 - 01:35 PM in Molecular Cloning

I guess you use Calcium Chlorid and using all tranformed E. coli to plated on Amp plates.



#147972 Difficult Ligation - Details Inside

Posted by Pangea on 13 January 2013 - 12:33 PM in Molecular Cloning

XbaI is dam sensitive. Spe1 is compatible with Xba1. Maybe, use klenow and ligate blunt end. Did you do Re-cutting? PCR with Taq or Pfu? Generally, try different ratio for ligation.



#147977 Difficult Ligation - Details Inside

Posted by Pangea on 13 January 2013 - 01:28 PM in Molecular Cloning

Better to increase insert. On your Gel you dont see your expected size? Maybe difficult on this size of an insert. With recutting i mean where do you no that its not working?



#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.



#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?



#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?



#147549 Large-Scale Purification of Non-Secreted Protein in E.Coli or CHO Cells

Posted by Pangea on 05 January 2013 - 03:38 PM in Protein Expression and Purification

How can you lyse the cells if you have 1000 l or more? And purify from a Bioreactor?



#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.



#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.



#147495 Replication of the Plasmid DNA

Posted by Pangea on 05 January 2013 - 01:01 AM in Genetics and Genomics

My Question would be which enzyme do we need in replicating Plasmids in E.coli? Any idea.



#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.



#147498 Questions about proper technical approach

Posted by Pangea on 05 January 2013 - 01:32 AM in Molecular Biology

Maybe you can check first whether it is a gene or not by using biotool like finding ORF, start and stop codons etc. Than you should clone this region if possible with this promotor.



#147548 Replication of the Plasmid DNA

Posted by Pangea on 05 January 2013 - 03:25 PM in Genetics and Genomics

You mean the Replication machinary.



#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?



#147665 Replication of the Plasmid DNA

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

:)




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