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Pangea's Content

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



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#147380 Residues on the N terminal GST Tag

Posted by Pangea on 03 January 2013 - 09:10 AM in Molecular Cloning

İ have a stupid question. İf i clone a HİS tag directly to my rProtein İ have no Amino Acid inbetween. But in case of GST Tag İ am getting additionally AA to my rProtein. Am İ right? How can İ avoid this.

Best regards



#147382 promoter question

Posted by Pangea on 03 January 2013 - 09:29 AM in -Microbiology and Virology-

The Lac Operon can be on the Vector itself or in the Strains Genome. Lac-Promotor is just to help RNA Polymerase to find his way.



#147386 GST TAG

Posted by Pangea on 03 January 2013 - 09:56 AM in Molecular Cloning

Lets say İ would like to clone my Gene BamHI and Not1 into pGEX GST N. When İ do that i will get additional Amino acids on my rProtein after cleavge with thrombin. İ think gly will not effect my overall structure but nevertheless its there.



#147387 Avoiding PTM Actylation of rProtein

Posted by Pangea on 03 January 2013 - 10:00 AM in Protein and Proteomics

How can İ prevent acylation (Lys) of my recombinant protein in E. coli or Mammalian cells?



#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



#147389 Plasmides run higher than marker

Posted by Pangea on 03 January 2013 - 10:14 AM in Electrophoresis

Heat up your Marker and cool it down. For the first time take an aliquot and heat up 94c. Use it look what happens.



#147390 GST- tagged protein expressed in insoluble

Posted by Pangea on 03 January 2013 - 10:46 AM in Protein Expression and Purification

SDS-Page check. İn Frame check.



#147427 GST TAG

Posted by Pangea on 04 January 2013 - 05:21 AM in Molecular Cloning

It is a common problem, the only real solution is to PCR a short tag (e.g. FLAG, HA) onto your DNA sequence and use that instead of the GFP

its not GFP its GST maybe it doesnt matter. Do you have source read more about it???



#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.



#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.



#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.



#147434 Questions about proper technical approach

Posted by Pangea on 04 January 2013 - 05:50 AM in Molecular Biology

Maybe you have to introduce this region upstream to a mammalian expression vector. Cut and Paste the new Promotor. Little bit tricky would be to use recombination and deleting the region.



#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.



#147447 siRNA in E.Coli

Posted by Pangea on 04 January 2013 - 10:23 AM in siRNA, microRNA and RNAi

Do E.coli have siRNA Metabolism?



#147469 protein concentration and Pull down

Posted by Pangea on 04 January 2013 - 12:43 PM in Biochemistry

Check the manual for the capacity of magnatic beads? If they are saturated its no matter how much you start with.



#147472 DNA extraction - PCR Problem

Posted by Pangea on 04 January 2013 - 12:52 PM in PCR, RT-PCR and Real-Time PCR

You must have a isolation protocol, as far i know if you have good primers ploidity should not be a problem. You are not the first person working with. It tells you just the copy number of identical chromosomes. Isolated DNA should be clean.



#147477 Break Gram-Negative Bacteria

Posted by Pangea on 04 January 2013 - 02:25 PM in Microbiology

What is the best method to break E.coli ? And get rid of DNA ?



#147480 How to transform adherent cells to suspension (CHO cells)

Posted by Pangea on 04 January 2013 - 02:45 PM in Tissue and Cell Culture

I want generate adherent cells to suspension cells. I not mean trypsinyzing. Can anyone help?



#147481 TRYPSIN

Posted by Pangea on 04 January 2013 - 02:51 PM in Molecular Cloning

How can i produce Cell culture Trypsin? Any Idea?



#147482 CIAP dilution

Posted by Pangea on 04 January 2013 - 03:12 PM in Molecular Cloning

1 Unit means 1 microMol Substrate in 60 minutes processing by the Enzyme (Enzyme Depending).



#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



#147493 TRYPSIN

Posted by Pangea on 05 January 2013 - 12:52 AM in Molecular Cloning

To make it more cheaper ;). Dou you have an idea? Maybe i will get the clone from IMAGE Consortium. Or get the trypsin from slaughterhouse.



#147494 siRNA in E.Coli

Posted by Pangea on 05 January 2013 - 12:57 AM in siRNA, microRNA and RNAi

Sounds good.Thanks.



#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.



#147496 protein concentration and Pull down

Posted by Pangea on 05 January 2013 - 01:10 AM in Biochemistry

No, of course if you use more magnetic beats you will increase you yield depending on your experiment design. But as I said you beads have capacity ana useing a lot of beats are not recommend. But in different concentration as you said,




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