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Phage Display [View Printable]
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TaqMan
Group: Member
Posts: 13
Joined: Jun 29, 2005
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Anyone here is working with Phage Display technology ??? |
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 Posted Jun 29, 2005, 17:26 PM |
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Carson O Genic
Group: Member
Posts: 152
Joined: Jun 22, 2005
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Yes...?
Edit post admonishment: Yes, I've used phage display technology to generate antibodies recognizing antigens on human blood cells. I beleive there is great promise for the technology, but we've also had our share of problems along the way. Is there a particular aspect of phage display technology that interests you? |
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| Posted Jul 01, 2005, 2:22 AM |
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Soudabeh
Group: Member
Posts: 256
Joined: Apr 23, 2004
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| Carson O'Genic said: | | Yes...? |
Dear member, Please reply with a full sentence and explain what kind of work you are doing. Yes or no replies are not considered scientific responses. Please communicate your information to your fellow members in full. Thanks, Scientist Solutions, Inc. |
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| Posted Jul 01, 2005, 3:23 AM |
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ding
Group: Member
Posts: 27
Joined: Jul 26, 2005
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| TaqMan said: | | Anyone here is working with Phage Display technology ??? |
I am working with a phage display library for scFv. I'm just a beginner and would very much appreciate to get hints on the solution to my problems and provide my opinion on any issue being posted here. |
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| Posted Jul 29, 2005, 13:57 PM |
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Carson O Genic
Group: Member
Posts: 152
Joined: Jun 22, 2005
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Ding,
Welcome, and please fell free to post your questions and opinions. If I can answer any questions I will. |
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| Posted Jul 29, 2005, 14:16 PM |
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TaqMan
Group: Member
Posts: 13
Joined: Jun 29, 2005
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Im just a beginner too...im working with scFv too..... |
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| Posted Jul 29, 2005, 19:13 PM |
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ding
Group: Member
Posts: 27
Joined: Jul 26, 2005
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| TaqMan said: | | Im just a beginner too...im working with scFv too..... |
1) when amplifying the library is it better to start from glycerol stocks (i.e. bacteria carrying the library phagemids, possibly those resulting from the original ligation reaction) or I may start from a pre-existing phage stock with which I infect naive bacteria cells (couldn't this cause a loss in the new stocks' representativity of the original library)? |
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| Posted Jul 30, 2005, 16:19 PM |
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ding
Group: Member
Posts: 27
Joined: Jul 26, 2005
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| Carson O'Genic said: | Ding,
Welcome, and please fell free to post your questions and opinions. If I can answer any questions I will. |
| TaqMan said: | | Im just a beginner too...im working with scFv too..... |
2) the new phage stocks obtained after amplification are better stored in PBS (or similar buffer) at -20°C or PBS-glycerol at -70°C? Which way is better to preserve from proteolysis and to guarantee best conditions at the time of selection (panning)? ... may glycerol interfere? |
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| Posted Jul 30, 2005, 16:21 PM |
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ding
Group: Member
Posts: 27
Joined: Jul 26, 2005
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| Carson O'Genic said: | Ding,
Welcome, and please fell free to post your questions and opinions. If I can answer any questions I will. |
| TaqMan said: | | Im just a beginner too...im working with scFv too..... |
3) (this is a question I have already posted in the microbiology section) I'm using TG1 E. coli. To preserve the F' pilus episome (necessary for infection by M13 phage) it is advisable to start coltures picking single colonies from a streak on minimal-medium-plates. I'm having troubles in finding the exact composition for the minimal medium, since I don't know if any vitamin/amminoacid must be supplemented to the salts and carbon source (M9 salts, MgSO4, CaCl2, glucose - as from the recipe on Sambrook et al.). Yesterday I started TG1 liquid coltures in 2XTY (complete medium) and minimal medium (Sambrook composition) and this morning I found the 2XTY colture fully grown and a poorly grown (yet growing) minimal colture: I wonder if it's normal (and acceptable for future infection) that bacteria grow less in a poorer (minimal) medium or if the minimal medium that I used lacks any supplement. Would you be able to provide me with a precise composition/recipe of the minimal medium that I'd better employ? THANK YOU ALL! |
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| Posted Jul 30, 2005, 16:29 PM |
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Soudabeh
Group: Member
Posts: 256
Joined: Apr 23, 2004
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| ding said: | | Carson O'Genic said: | Ding,
Welcome, and please fell free to post your questions and opinions. If I can answer any questions I will. |
| TaqMan said: | | Im just a beginner too...im working with scFv too..... |
3) (this is a question I have already posted in the microbiology section) I'm using TG1 E. coli. To preserve the F' pilus episome (necessary for infection by M13 phage) it is advisable to start coltures picking single colonies from a streak on minimal-medium-plates. I'm having troubles in finding the exact composition for the minimal medium, since I don't know if any vitamin/amminoacid must be supplemented to the salts and carbon source (M9 salts, MgSO4, CaCl2, glucose - as from the recipe on Sambrook et al.). Yesterday I started TG1 liquid coltures in 2XTY (complete medium) and minimal medium (Sambrook composition) and this morning I found the 2XTY colture fully grown and a poorly grown (yet growing) minimal colture: I wonder if it's normal (and acceptable for future infection) that bacteria grow less in a poorer (minimal) medium or if the minimal medium that I used lacks any supplement. Would you be able to provide me with a precise composition/recipe of the minimal medium that I'd better employ?
THANK YOU ALL! |
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| Posted Aug 02, 2005, 16:57 PM |
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TaqMan
Group: Member
Posts: 13
Joined: Jun 29, 2005
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The problems Im have are different.... Im using Barbas protocol to clone ScFv fragments into XL1-Blue electrocompetents cells, but my digestions arent good. After the digestion, when I make a electroforesis, the collum shows me 4 bands,single-cut plasmid, other is the double-cut plasmid (a weak band), a unknow band and the Stuffer band (region that is removed from the plasmid). In size order.
I have changed ALL the materials, but the results are the same... It could not be my mistake because my friend tryed too and encounter the same results....
What was the expected result was just 2 bands only, the double-cut plasmid and the stuffer.
If someone could give me some help I would appreciate :) |
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| Posted Aug 03, 2005, 9:14 AM |
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ding
Group: Member
Posts: 27
Joined: Jul 26, 2005
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| TaqMan said: | The problems Im have are different.... Im using Barbas protocol to clone ScFv fragments into XL1-Blue electrocompetents cells, but my digestions arent good. After the digestion, when I make a electroforesis, the collum shows me 4 bands,single-cut plasmid, other is the double-cut plasmid (a weak band), a unknow band and the Stuffer band (region that is removed from the plasmid). In size order.
I have changed ALL the materials, but the results are the same... It could not be my mistake because my friend tryed too and encounter the same results....
What was the expected result was just 2 bands only, the double-cut plasmid and the stuffer.
If someone could give me some help I would appreciate :) |
I have bought Barba's "Phage display - a lab manual" (though I do not have it here with me now). I don't understand whether you are making a new library (trying to clone fragments coding for a variety of scFv) or you have already selected a phage clone whose scFv you now need to subclone into a different plasmid. Anyway, yours seems to be a common (not always esy-to-solve) problem of molecular cloning. The unknown band that you find in the four-band pattern after digestion might be the undigested circular plasmid, which always migrates faster than the linearized (single-cut) plasmid and than the linear double-cut plasmid (provided the insert - stuffer - that you remove by digestion is not a huge part of the whole plasmid construct). Of course, the migration of the unknown band should be consistent with the size of the whole plasmid: according to my experience an uncut plasmid of around 4500 bp migrates as a linear dsDNA between 2 and 2.5 Kbp. The problem, as you outline it, is one of poor digestion efficiency and that may be due to the quality of your plasmid DNA (try use a high- purity extraction kit like Qiagen or similar or change the E.coli strain that carries your plasmid and from which you made a prep) or the restriction enzyme you're using is deteriorated or not enough concentrated in the reaction (is the buffer ok? if using two different enzymes I suggest never performing a double-digest, but rather two sequential digestions, especially if the buffers recommended for the two enzymes are different). Another possibility is that the restriction enzymes are methylation-sensitive and that their recognition sequences are methilated in your plasmid DNA (but that's hardly probable if you're exactly following a well-tested protocol from a manual). To solve or at least try to face this kind of problems have a look at some molecular cloning manual, even catalogs of some molecular biology company can be of use, for example New England Biolabs. Or you may simply post your topic in the molecular biology section ... good luck! SOMEONE PLEASE HELP ME WITH THE QUESTIONS POSTED ABOVE!!! |
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| Posted Aug 04, 2005, 19:29 PM |
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ding
Group: Member
Posts: 27
Joined: Jul 26, 2005
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what is the expected phage titer (tu/mL) after you amplify a library from a 500 mL culture of phagemid-carrying bacteria and resuspend the PEG-precipitated phage-particles in 10 mL volume? |
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| Posted Aug 12, 2005, 15:33 PM |
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ding
Group: Member
Posts: 27
Joined: Jul 26, 2005
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I have tested selected phage clones by ELISA. All of them are positive ... but an unrelated phage clone is positive as well ... The phage particles from each clone have been 5 to 15-fold concentrated from culture medium by PEG-precipitation because a previous attempt with medium surnatant used "as is" in ELISA gave poor results for well-characterized positive phage clones. I wonder if the residual PEG in my phage preparation may make the phage "sticky" and favour aspecific binding of the phage particles to the ELISA wells with occurrence of falsely positive reactions. Anyone has any experience of this kind? |
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| Posted Aug 19, 2005, 18:03 PM |
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ding
Group: Member
Posts: 27
Joined: Jul 26, 2005
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The titers of 3 pannings of a scFv phage display library against two different proteins:
protein A: pan1 2*10e5 / pan2 2*10e5 / pan3 8*10e5
protein B: pan1 5*10e4 / pan2 10e6 / pan3 10e7
what would you do? |
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| Posted Sep 13, 2005, 20:42 PM |
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