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    <title>Discussion forums</title>
    <link>http://biowww.net/forum/index</link>
    <description><![CDATA[]]></description>
    <language>EN</language>
    <pubDate>Fri, 25 Apr 2008 15:10:33 -0500</pubDate>
    <lastBuildDate>Fri, 25 Apr 2008 15:10:33 -0500</lastBuildDate>
    <category>Discussion forums</category>
    <generator>Phorum 5.0.13a</generator>
    <ttl>600</ttl>
    <item>
      <title>[Proteomics and protein biochemistry forum] Ratio of stacking and separating gel</title>
      <link>http://biowww.net/forum/read/18/2508/2508#msg-2508</link>
      <author>Tiffy</author>
      <description><![CDATA[Hello everybody,
My problem is that I have a large volume of a protein sample. But my capacity of the gel pockets are restricted. Now my question can I pour a gel with a long stacking gel that my volume will fit properly? Because I don`t want to precipitate my samples. Or will I get problems? I am not sure. Once I read one should have a long separating gel. My mw of my protein is about 180kDa. 
Please help me.
Thanks 
Tiffy]]></description>
      <category>Proteomics and protein biochemistry forum</category>
      <guid isPermaLink="true">http://biowww.net/forum/read/18/2508/2508#msg-2508</guid>
      <pubDate>Fri, 25 Apr 2008 15:10:33 -0500</pubDate>
    </item>
    <item>
      <title>[Proteomics and protein biochemistry forum] Re: looking for protocol for hollow fiber cleaning</title>
      <link>http://biowww.net/forum/read/18/2378/2507#msg-2507</link>
      <author>Lalith</author>
      <description><![CDATA[We are using alkaline cleaning (with Cl) and then acidic cleaning.  We purchace our cleaning chemicals from Jonson Diversy.  Our process is protein purification so this cleaning chemicals works nicely with us.  I think the cleaning protocol should deside according to your process and therefore when you try the above method sometimes it may not work as well.  But any way above method can work in a very broad range. Please let me know if you need any further assistance in this regard. my email lalith@samartex.com]]></description>
      <category>Proteomics and protein biochemistry forum</category>
      <guid isPermaLink="true">http://biowww.net/forum/read/18/2378/2507#msg-2507</guid>
      <pubDate>Wed, 23 Apr 2008 08:55:45 -0500</pubDate>
    </item>
    <item>
      <title>[Molecular biology troubleshooting forum] compatibility vector/competent cell</title>
      <link>http://biowww.net/forum/read/5/2506/2506#msg-2506</link>
      <author>frantzisca</author>
      <description><![CDATA[Hi,
could someone tell me something about possible incompatibility between some kind of vector and competent cells?
I'm working with PBluescript KS+ (2,9 kb) containing a 6,5 kb DNA fragment, and I'm trying to ligate in it a 4,7 kb fragment. When I transform with OneShot TOP10 electrocomp E.Coli cells (following protocol conditions), the next day I find every time a patina over the entire plate, and any colonies, sometime 1-2, but negative.
It is not contamination nor wrong Amp because I did the experiment by using the same stock of LB Amp plates but with different kind of cells (XL1Blue and TOP10 both chemically competents) and I never found this patina. I suspect some kind of incompatibility between the PBluescript (with a so large foreign DNA) and the OneShot TOP10 electrocomp E.Coli cells. I cannot find other explanation.
Could you help me please?

Frantzisca]]></description>
      <category>Molecular biology troubleshooting forum</category>
      <guid isPermaLink="true">http://biowww.net/forum/read/5/2506/2506#msg-2506</guid>
      <pubDate>Mon, 21 Apr 2008 10:17:49 -0500</pubDate>
    </item>
    <item>
      <title>[Proteomics and protein biochemistry forum] optimizing protein expression in BL21(AI)-adjust arabinose % 1st or IPTG1st?</title>
      <link>http://biowww.net/forum/read/18/2505/2505#msg-2505</link>
      <author>doremi_luv</author>
      <description><![CDATA[Hi,

Currently i'm using the bl21(AI) cell line to express my protein. I'm still at the beginning stage whereby i optimize the % of arabinose and concentration of IPTG.

What i'd like to know is, should i optimise the % arabinose first, and then followed by IPTG?
or should i optimise the IPTG first, followed by % arabinose?

THanks for any help in advance!!!]]></description>
      <category>Proteomics and protein biochemistry forum</category>
      <guid isPermaLink="true">http://biowww.net/forum/read/18/2505/2505#msg-2505</guid>
      <pubDate>Mon, 21 Apr 2008 02:37:40 -0500</pubDate>
    </item>
    <item>
      <title>[Bioinformatics forum and biostatistics forum] Training cum Recruitment in GVK Bio Informatics division on computational drug design</title>
      <link>http://biowww.net/forum/read/22/2503/2503#msg-2503</link>
      <author>biocampus</author>
      <description><![CDATA[Dear All
GVK Bio-The No.1 Contract Research Organization in India has launched its new branch of bioCampus in Chennai Informatics division offering &quot;Protein modeling and Drug Design&quot;-training cum recruitment program and projects for final year M.Sc, B.Tech, M.Tech, B.Pham, M.Pham students. More than 500 students have been recruited in our Informatics division from bioCampus.

Mail us for any further clarifications.

bioCampus
GVK Biosciences Private Limited,
#37, Sterling Road, Nungambakkam,
Chennai-600034, India
T +91 044 - 66293000, 66293007
F +91 44 66293199
Mail to biocampus.chennai@gvkbio.com
Web: www.bio-campus.com

Warm Regards, 

Team bioCampus
]]></description>
      <category>Bioinformatics forum and biostatistics forum</category>
      <guid isPermaLink="true">http://biowww.net/forum/read/22/2503/2503#msg-2503</guid>
      <pubDate>Fri, 18 Apr 2008 03:32:13 -0500</pubDate>
    </item>
    <item>
      <title>[Proteomics and protein biochemistry forum] Finding Secondary Structure of a Protein</title>
      <link>http://biowww.net/forum/read/18/2501/2501#msg-2501</link>
      <author>nhabibi</author>
      <description><![CDATA[Hi everybody,

I want to write a program to predict the contact map of proteins. It uses some features as Neural Network input. One of them is Secondary structure. I know that a popular method is DSSP. I got the previously calculated dssp file for some proteins form the internet, but they don't match with their pdb files. for example you can compare the 1a1v.pdb and 1a1v.dssp, the dssp file is longer!
Another way, I think, is to use the dssp program directly, but I can't fine a free version on th internet. Can anybody help me? I really need it. glare.gif

Thanks ]]></description>
      <category>Proteomics and protein biochemistry forum</category>
      <guid isPermaLink="true">http://biowww.net/forum/read/18/2501/2501#msg-2501</guid>
      <pubDate>Thu, 17 Apr 2008 12:41:39 -0500</pubDate>
    </item>
    <item>
      <title>[Cell biology and histology methods forum] Oligomycin B </title>
      <link>http://biowww.net/forum/read/19/2500/2500#msg-2500</link>
      <author>Tiffy</author>
      <description><![CDATA[Hello everybody,

 I have a question about ATP depletion. I have seeded my tumor cell line on glas coverslips and treated them with oligomycin B (50µM, 4h). Before I have started my experiment I removed the medium with glucose and replaced it with glucose-free medium. After the incubation time and before fixation I wanted to wash my cells mit PBS. Unfortunately I washed all cells away.  I could fix just a few cells. Okay, then I reduced the concentration 5µM for an hour. The same result. 
Who knows a concentration of oligomycin b for MCF-7? It is very urgent. 
I am grateful for all comments.
Thanks in advance.
Tiffy]]></description>
      <category>Cell biology and histology methods forum</category>
      <guid isPermaLink="true">http://biowww.net/forum/read/19/2500/2500#msg-2500</guid>
      <pubDate>Wed, 16 Apr 2008 12:55:04 -0500</pubDate>
    </item>
    <item>
      <title>[Molecular biology troubleshooting forum] DNA precipitation by ethanol.</title>
      <link>http://biowww.net/forum/read/5/2498/2498#msg-2498</link>
      <author>Shivani</author>
      <description><![CDATA[hello everyone! I have been isolating DNA from blood using high salt precipitation method by NaCl. From past few months, at final step of DNA precipitation by ethanol I get DNA strands only in few tubes and not in all. Although when I add ethanol i can see some colloidal part but it gets dissolved completely before actually giving the DNA strands. 
Is there any method by which I can precipitate DNA from this solution??? or is there any alternative to this step? my samples are very crucial, can not afford to lose more...please suggest]]></description>
      <category>Molecular biology troubleshooting forum</category>
      <guid isPermaLink="true">http://biowww.net/forum/read/5/2498/2498#msg-2498</guid>
      <pubDate>Mon, 14 Apr 2008 12:37:35 -0500</pubDate>
    </item>
    <item>
      <title>[Molecular biology troubleshooting forum] Re: Direct PCR from blood</title>
      <link>http://biowww.net/forum/read/5/2473/2496#msg-2496</link>
      <author>elm3r</author>
      <description><![CDATA[there seems to be something in ScienceDirect:

http://www.sciencedirect.com/science?_ob=ArticleURL&amp;_udi=B6T30-3SY3GVJ-3&amp;_user=949111&amp;_rdoc=1&amp;_fmt=&amp;_orig=search&amp;_sort=d&amp;view=c&amp;_acct=C000049116&amp;_version=1&amp;_urlVersion=0&amp;_userid=949111&amp;md5=56ad08514dc387cf42d6a6edc8608f3e

if you don't have a access, reply to this post to say it.

]]></description>
      <category>Molecular biology troubleshooting forum</category>
      <guid isPermaLink="true">http://biowww.net/forum/read/5/2473/2496#msg-2496</guid>
      <pubDate>Fri, 11 Apr 2008 08:49:56 -0500</pubDate>
    </item>
    <item>
      <title>[Molecular biology troubleshooting forum] Re: cells that do not contain DNA..</title>
      <link>http://biowww.net/forum/read/5/2493/2495#msg-2495</link>
      <author>elm3r</author>
      <description><![CDATA[you have a reply there:

http://www.physicsforums.com/showthread.php?t=226652

...

hum... plus there might be something in the amniotic fluids... but I don't know much more...]]></description>
      <category>Molecular biology troubleshooting forum</category>
      <guid isPermaLink="true">http://biowww.net/forum/read/5/2493/2495#msg-2495</guid>
      <pubDate>Fri, 11 Apr 2008 08:38:04 -0500</pubDate>
    </item>
    <item>
      <title>[Molecular biology troubleshooting forum] Re: TOPO TA cloning problem</title>
      <link>http://biowww.net/forum/read/5/2357/2494#msg-2494</link>
      <author>elm3r</author>
      <description><![CDATA[if you want to purify 2ug of DNA from a gel slice on a Quiagen colum, you are going to end up with 200ng, my own experience ! Always use the TE buffer to elute and start with at least 5 times more DNA than you need for downstream steps...

about the cloning, this TOPO TA cloning thing is funny, it's a complete mess and a joke, it actually mixes the TOPO system found in some Gateway entry vector, so that you can clone directionally your PCR products with the help of one base-pairing site and Topoisomerases... The TA cloning is classical cloning with T overhangs, meaning that you can ONLY clone PCR products that have been generated with Taq-related polymerases... (they add A overhangs to the PCR product)...

This is where TOPO TA sucks : you need to have A overhangs on your PCR product otherwise it won't fit in the vector, it's just to &quot;simplify&quot; of the user by making him pay much more money.

anyway, as I like to say : The only thing that Eppendorf do well is their Eppendorf Tubes, and 30% of the money going in research actually goes to Invitrogen...

thiefs !

:P]]></description>
      <category>Molecular biology troubleshooting forum</category>
      <guid isPermaLink="true">http://biowww.net/forum/read/5/2357/2494#msg-2494</guid>
      <pubDate>Fri, 11 Apr 2008 08:28:26 -0500</pubDate>
    </item>
    <item>
      <title>[Molecular biology troubleshooting forum] cells that do not contain DNA..</title>
      <link>http://biowww.net/forum/read/5/2493/2493#msg-2493</link>
      <author>soidal</author>
      <description><![CDATA[Hi,

i would so much appreciate a list of anucleate cells in human body.
i know platelets and red blood cells are anucleate.
Are there more?

Thanks a million.]]></description>
      <category>Molecular biology troubleshooting forum</category>
      <guid isPermaLink="true">http://biowww.net/forum/read/5/2493/2493#msg-2493</guid>
      <pubDate>Sat, 05 Apr 2008 00:07:05 -0500</pubDate>
    </item>
    <item>
      <title>[Molecular biology troubleshooting forum] Re: TOPO TA cloning problem</title>
      <link>http://biowww.net/forum/read/5/2357/2491#msg-2491</link>
      <author>drsgarrett</author>
      <description><![CDATA[Which gel purification kit are you using?  I find the Qiaex gives crappy yields always.  Quiagen's column purification works much better.  Are you eluting in the given elution  buffer or water?  If your water's pH is wrong, you won't get anything off the column.  In my experience, the EB buffer does not give problems in most downstream reaction.s]]></description>
      <category>Molecular biology troubleshooting forum</category>
      <guid isPermaLink="true">http://biowww.net/forum/read/5/2357/2491#msg-2491</guid>
      <pubDate>Fri, 04 Apr 2008 15:38:58 -0500</pubDate>
    </item>
    <item>
      <title>[Molecular biology troubleshooting forum] Re: chIP problem</title>
      <link>http://biowww.net/forum/read/5/2418/2490#msg-2490</link>
      <author>drsgarrett</author>
      <description><![CDATA[Are A and B the December ones that worked?

If it worked one month and then didn't the next month, my best bet is that some of your reagents went off.  If you're reasonably certain the antibodies are fine, I'd remake your buffers (assuming you aren't using a kit) and see if that works with a known system before going back to your experiment of interest.

I doubt the sonication is an issue unless your sonicator broke.]]></description>
      <category>Molecular biology troubleshooting forum</category>
      <guid isPermaLink="true">http://biowww.net/forum/read/5/2418/2490#msg-2490</guid>
      <pubDate>Fri, 04 Apr 2008 15:36:52 -0500</pubDate>
    </item>
    <item>
      <title>[Molecular biology troubleshooting forum] Re: TOPO TA cloning</title>
      <link>http://biowww.net/forum/read/5/2464/2489#msg-2489</link>
      <author>drsgarrett</author>
      <description><![CDATA[I've recently cloned both a 4kb and a 5kb fragment into Topo as well as pGL vectors.  

First, are you gel purifying your PCR products?  If not, the issue is that you have multiple products from early termination and such, and the TOPO is preferring the smaller ones.  Do enough PCR so you can do several lanes so you'll get back enough of the product to do the transformation with.

Second, use all of your cloning reaction mixture in one of your transformation conditions.  Leave the cloning reaction on the bench for the longest time suggested by the book (up to 20 minutes I think), then put into your compentent cells and incubate on ice the full 30 minutes.  This maximizes the efficiency in my experience.

After that, screen, screen, and screen.  If you are only missin 800 bp, odds are, there are reactions with the full amount.  Doing the gel purification so that all your potential products are about the same size will maximize your number of positives.  

sg]]></description>
      <category>Molecular biology troubleshooting forum</category>
      <guid isPermaLink="true">http://biowww.net/forum/read/5/2464/2489#msg-2489</guid>
      <pubDate>Fri, 04 Apr 2008 15:33:18 -0500</pubDate>
    </item>
    <item>
      <title>[Molecular biology troubleshooting forum] Micro-injected marine collagen- in a lip plumper?</title>
      <link>http://biowww.net/forum/read/5/2488/2488#msg-2488</link>
      <author>classic3903</author>
      <description><![CDATA[Is Fusionbeauty Lipfusion really safe eg is it nano tech, etc? I asked the company 2x: Q1Have the Lipfusion ingredients been micronized or similar to your skin products that are ‘nano’? Its described as Tiny, dehydrated, marine collagen micro spheres. A1 Tiny, dehydrated, marine collagen microspheres are instantly absorbed by the lips, and seek out the body’s natural moisture. As soon as the spheres become re-hydrated, they plump and hold onto the moisture they’ve collected, creating a mild &quot;swelling &quot; of the lip tissue. The result is fuller, smoother, perfectly plumped lips in seconds. Results can last for up to 48 hours. Q2 I have noticed that a new face product of yours has the word nano in it -Is this used in your Lip Fusion plumper? A2 - The Nanospheres is the delivery system harnessed in Liftfusion Mtox Transdermal Face Lift. The word &quot;nano&quot;is for nanospheres these are particles that are even smaller than microspheres and release the Mtox formula in in order to actually absorb into the specific targeted areas upon contact. 

http://www.lipfusi on.com/?page=science 

Many products claim 'collagen' as a featured ingredient, when, in truth the collagen molecules are too large to penetrate the skin. LipFusion harnesses the clinically proven plumping and hydrating powers of new-patented marine collagen-filling spheres. This revolutionary technology dehydrates the collagen molecule into tiny microspheres in order to penetrate, or micro-inject, into the lips and then search for the body's natural water to re-hydrate, creating a beautifully natural, full-out pout. SO is it safe ... or true to its claims?  ]]></description>
      <category>Molecular biology troubleshooting forum</category>
      <guid isPermaLink="true">http://biowww.net/forum/read/5/2488/2488#msg-2488</guid>
      <pubDate>Thu, 03 Apr 2008 16:21:09 -0500</pubDate>
    </item>
    <item>
      <title>[Pharmacology and toxicology forum] Micro-injected marine collagen- in a lip plumper?</title>
      <link>http://biowww.net/forum/read/29/2487/2487#msg-2487</link>
      <author>classic3903</author>
      <description><![CDATA[Micro-injected marine collagen- in a lip plumper?

Is Fusionbeauty Lipfusion really safe eg is it nano tech, etc? I asked the company 2x: Q1Have the Lipfusion ingredients been micronized or similar to your skin products that are ‘nano’? Its described as Tiny, dehydrated, marine collagen micro spheres. A1 Tiny, dehydrated, marine collagen microspheres are instantly absorbed by the lips, and seek out the body’s natural moisture. As soon as the spheres become re-hydrated, they plump and hold onto the moisture they’ve collected, creating a mild &quot;swelling &quot; of the lip tissue. The result is fuller, smoother, perfectly plumped lips in seconds. Results can last for up to 48 hours. Q2 I have noticed that a new face product of yours has the word nano in it -Is this used in your Lip Fusion plumper? A2 - The Nanospheres is the delivery system harnessed in Liftfusion Mtox Transdermal Face Lift. The word &quot;nano&quot;is for nanospheres these are particles that are even smaller than microspheres and release the Mtox formula in in order to actually absorb into the specific targeted areas upon contact. http://www.lipfusi on.com/?page=scien ce Many products claim 'collagen' as a featured ingredient, when, in truth the collagen molecules are too large to penetrate the skin. LipFusion harnesses the clinically proven plumping and hydrating powers of new-patented marine collagen-filling spheres. This revolutionary technology dehydrates the collagen molecule into tiny microspheres in order to penetrate, or micro-inject, into the lips and then search for the body's natural water to re-hydrate, creating a beautifully natural, full-out pout. SO is it safe ... or true to its claims?  ]]></description>
      <category>Pharmacology and toxicology forum</category>
      <guid isPermaLink="true">http://biowww.net/forum/read/29/2487/2487#msg-2487</guid>
      <pubDate>Thu, 03 Apr 2008 16:19:06 -0500</pubDate>
    </item>
    <item>
      <title>[Cell biology and histology methods forum] Re: cultivation of hybridomas</title>
      <link>http://biowww.net/forum/read/19/2183/2486#msg-2486</link>
      <author>aphio-igor</author>
      <description><![CDATA[It depends on the hybridoma, but most that I've dealt with are in suspension.  As a general 'most are this way', your target range of concentration is 2e5 to 2e6 cells per mL, with most lines preferring 3e5 to 1e6.  As for the media choice, again, it depends on the cells themselves, but most Balb/c based hybridomas I've worked with or developed have preferred DMEM as their basal constituent, adding 3% &quot;LNN&quot; (that is, 1% each of: 200mM L-Glutamine, MEM Non- essential Amino Acids, and NaPyruvate) per litre.  10% FBS is standard, and sometimes gentamycin is used as an antibiotic/ antimycotic (personal preference is to omit it, as it allows contaminant detection and containment as soon as it happens).  

Again, that's the case for **most** hybridoma lines, and shouldn't be used exclusively as a hard and fast rule.  If you purchased the cells from a provider, might I ask what the cell line's name is?  I might be able to provide more help with that.

Beyond that, I'll restate what was said before, in that the T- flasks should have a slanted neck leading to the cap.  You want the flask's two largest sides laid parallel to to the incubator's shelf, with the neck slanted slightly up.  If the cap has vents for the gas exchange for CO2, you're fine, but if not, don't remember to loosen the cap a tad once in the incubator.

I hope that this helps, and feel free to ask any questions... it may take a while to reply, as I have a newborn at home and I'm the &quot;official diaper changer&quot; ;)
Kind regards,
Pete Gramp (aphio-igor)]]></description>
      <category>Cell biology and histology methods forum</category>
      <guid isPermaLink="true">http://biowww.net/forum/read/19/2183/2486#msg-2486</guid>
      <pubDate>Wed, 02 Apr 2008 14:15:54 -0500</pubDate>
    </item>
    <item>
      <title>[Cell biology and histology methods forum] Re: monkey keratinocytes</title>
      <link>http://biowww.net/forum/read/19/2323/2485#msg-2485</link>
      <author>aphio-igor</author>
      <description><![CDATA[I've heard of, though never worked with, a line from cynomolgus monkey skin... CYNOM-KI... though I think it has a fibroblastic morphology.  

hmm, a CABRI search turned up that ECACC offers the line under Catalog # 90071809, but again, I have no clue regarding the keratinocyte aspect - i do know it is from skin explant, but beyond that I am clueless.

Still, it may be what you're after... 

Actually, per subsection [0078] of http://www.wipo.int/pctdb/en/wo.jsp?ELEMENT_SET=F&amp;LANGUAGE=ENG&amp;KEY=06%2F082515&amp;IA=06%2F082515&amp;DISPLAY=DESC
(sorry for the long link) it IS, in fact, a &quot;cynomolgus monkey keratinocyte cell line&quot;.

Best regards,
Pete Gramp
(aphio-igor)]]></description>
      <category>Cell biology and histology methods forum</category>
      <guid isPermaLink="true">http://biowww.net/forum/read/19/2323/2485#msg-2485</guid>
      <pubDate>Wed, 02 Apr 2008 13:58:47 -0500</pubDate>
    </item>
    <item>
      <title>[Cell biology and histology methods forum] Re: CHO-S cell</title>
      <link>http://biowww.net/forum/read/19/2379/2484#msg-2484</link>
      <author>aphio-igor</author>
      <description><![CDATA[Hi, VPIlabs-
This may be a silly question, but when transfecting, which transfectant agent (chemical, kit, product name, etc) do you use?  The reason I ask is that some products function via a 'superglue- like' method (i.e. Polybrene, DEAE-Dextran derivitives, etc - mostly intended for viral vectors), other products work through membrane binding and engulfment, be it via lipid raft formation, receptor binding, or a myriad of other methods, and yet others function through membrane disruption and so- called &quot;weakening&quot;.  

Enough background rambling - the trypsin you use may be &quot;punching holes&quot; in the membrane and lysing the cells you want.  Why does the trypsin only affect the transfectants and not the residual parental, untransfected cells?  The transfecting agent/ product may be (emphasis on MAY be) only affecting those cells that get the plasmid - kind of makes sense, as the non- transfectants didn't get transfected for the same reason that the others did, that is, through action of the agent.  Bottom line is, the transfectants may have &quot;weakened&quot; membranes already, to get the plasmid punched through.  The trypsin, by chomping up the extracellular proteins, may induce a ton of &quot;bad ju- ju&quot; (technical jargon, I know...), including but not limited to apoptosis, etc.

How to solve it:  

First, you can spin out the trypsin from the trypsinized cells, then resuspend the cell pack/ pellet in fresh media.  Remember, trypsin isn't used up during the debridement process, so it will keep chewing those cells' proteins until something else stops it.  Serum has anti-trypsins, as well as tons of BSA (bovine serum albumin) which essentially dilutes out the cells' proteins from the total pool of proteins available for the trypsin to chew on.  As you're using Serum- Free media, I'm guessing the trypsin (while diluted out via fresh media) may not be fully inactivated when you initiate your suspension culture.  Oh, and with the trypsin/ transfection being so close together, I'd limit the spin to 125 x g.  You can spin for 10 minutes if needed, but don't go nuts and squish them at 200 x g... sorry if rude, but I'm speaking from experience when I say &quot;If you find you've done all this work, and then get the cells squished, that really stinks&quot;

Second, you can wait more than 4 hours post- transfection before debridement/ trypsinization.  I doubt that's the issue, and depending on what receptor you're looking at, it may be counterproductive to wait more than 4 hours - I don't know.  What I can say is that the interim period between transfection and trypsinization will allow the cells to start producing proteins again, aiding in membrane stability.

Third, you can use a different (synthetic?) debridement agent.  Trypsin is great, don't get me wrong, and relatively cheap besides, but *as a last resort*, you can try something else - I'm a huge supporter of TrypLE Select, a Gibco/ Invitrogen product (no affiliation besides being a VERY happy user).  The TrypLE Select dilutes out with the addition of fluids (media).  There are a whole myriad of other products out there, and I can point you in their direction if requested.

As for the CHO-S option, you can use shake- flasks at ~110 to 140 rpm in place of stirrers.  Note that CHO cells (any type that I have dealt with, and - for better or worse - that's more than I'd care to admit) are known for being &quot;stagnant buggers&quot;... that is, you may need to spike them with HT supplement to get them kick- started into DNA replication again.  The... oh goodness... H = hypoxanthine? T= thymidine?  just guesses - I've got a 3-day-old son, so forgive my memory lapse... anyhow, that HT sup will provide the needed 'juice' for the DNA replication.  Also, CHO's tend to be slow in producing L-glutamine, in general.  It isn't like they're going to up and die on you, but a few mL's of L-glut are worth their weight in gold when trying to get &quot;happier&quot; cells.  So what I'm getting at is that the Cho-S cells would *likely* work, but the CHO-K1's are likely better for the transfection itself, and the subsequent morphologic change/ media- change is much less of a problem - lesser of two evils, if you will.  Personal opinion, and I'm sure that others will disagree wholeheartedly.  

As for growing CHO's in suspension without stirring, again, one can shake them with shake- flasks.  I suppose you could use T- flasks, and they should work just fine, but be prepared for clumpy cells the first several passages.  (it gets better as they adapt to the media)

As for cheaper media, (i.e. Ham's F12, F12-K, etc.), I really wouldn't risk it.  As with all of this above 'book' that I've written, it is just one guy's experiences and opinions... but saving a buck now isn't worth the zillion bucks later for having to repeat the whole darned thing again.  Just my opinion, sorry if it came out poorly.  To help with costs, my experience is that CD-CHO +HT is your best bet.  I don't know what receptor you're looking at, so it's hard to recommend media - you may get oddball results with some of the media's components - but *generally* speaking, the CD-CHO with HT is your best bet.  A close second is Mach1, but that's intended more as an antibody- producing medium.  Other than that, unless I've done it wrong several times over (and trust me, that wouldn't be too far of a stretch, nor would it be the first time!), Ham's F12 won't be enough for your cells in a serum- free environment.  They'll be there, and likely some will be alive, but when transfecting and looking to select the clonal population, I'd sooner hedge my bets with a different media than risk all to save a few bucks - sorry again if that was worded poorly, but I'm just trying to save you grief in the long- run.

Speaking of grief in the long- run, our newborn has &quot;volunteered&quot; me for diaper duty... oh the joys...
Best of luck and kind regards,
Pete Gramp (aphio-igor)]]></description>
      <category>Cell biology and histology methods forum</category>
      <guid isPermaLink="true">http://biowww.net/forum/read/19/2379/2484#msg-2484</guid>
      <pubDate>Wed, 02 Apr 2008 12:55:03 -0500</pubDate>
    </item>
    <item>
      <title>[Cell biology and histology methods forum] CHO-K1 vs CHO-S cell</title>
      <link>http://biowww.net/forum/read/19/2483/2483#msg-2483</link>
      <author>VPIlabs</author>
      <description><![CDATA[Hi,

I have a problem concerning CHO-K1 cells.

I have been trying to transfect adherent CHO-K1 cells with multiple transfection agents, which worked. After that I'd like to dissociate these transfected cells to study internalisation of the transfected receptor (encoded on the plasmid) in suspension, but I have no surviving cells after this trypsinisation. I usually trypsinize them 4 hours after transfection and grow them in fresh medium till 24h post transfection. Is there anyone who has some suggestions or who has already done this?
I am able to keep non-transfected cells alive, so I think the combination of trypsinization and transfection just kills my cells.

Another option is to buy CHO-S cells wich are in suspension, but I have no stirrer inside our incubators. Is it possible to grow these cells without stirring them? Or will they start to adhere? Also, do you need the serum-free medium or can you use F12 or other (cheaper) media?

Thanks in advance for your reactions!]]></description>
      <category>Cell biology and histology methods forum</category>
      <guid isPermaLink="true">http://biowww.net/forum/read/19/2483/2483#msg-2483</guid>
      <pubDate>Wed, 02 Apr 2008 07:17:13 -0500</pubDate>
    </item>
    <item>
      <title>[Cell biology and histology methods forum] Re: CHO-S cell</title>
      <link>http://biowww.net/forum/read/19/2379/2482#msg-2482</link>
      <author>VPIlabs</author>
      <description><![CDATA[Hi,

I have a problem concerning CHO-S cells.

I have been trying to transfect adherent CHO-K1 cells with multiple transfection agents, which worked. After that I'd like to dissociate these transfected cells to study internalisation of the transfected receptor (encoded on the plasmid) in suspension, but I have no surviving cells after this trypsinisation. I usually trypsinize them 4 hours after transfection and grow them in fresh medium till 24h post transfection. Is there anyone who has some suggestions or who has already done this?
I am able to keep non-transfected cells alive, so I think the combination of trypsinization and transfection just kills my cells.

Another option is to buy CHO-S cells wich are in suspension, but I have no stirrer inside our incubators. Is it possible to grow these cells without stirring them? Or will they start to adhere? Also, do you need the serum-free medium or can you use F12 or other (cheaper) media?

Thanks in advance for your reactions!]]></description>
      <category>Cell biology and histology methods forum</category>
      <guid isPermaLink="true">http://biowww.net/forum/read/19/2379/2482#msg-2482</guid>
      <pubDate>Wed, 02 Apr 2008 07:12:54 -0500</pubDate>
    </item>
    <item>
      <title>[Bioinformatics forum and biostatistics forum] Re: looking for beta tester</title>
      <link>http://biowww.net/forum/read/22/2466/2481#msg-2481</link>
      <author>ete</author>
      <description><![CDATA[New update (some bugs fixed):

http://filebeam.com/6caec2b9d388c092eab6338533dc7807]]></description>
      <category>Bioinformatics forum and biostatistics forum</category>
      <guid isPermaLink="true">http://biowww.net/forum/read/22/2466/2481#msg-2481</guid>
      <pubDate>Tue, 25 Mar 2008 05:08:06 -0500</pubDate>
    </item>
    <item>
      <title>[Molecular biology troubleshooting forum] The Bioinformatica Solutions-training in bioinformatics</title>
      <link>http://biowww.net/forum/read/5/2480/2480#msg-2480</link>
      <author>shuchi</author>
      <description><![CDATA[Dear all,

The Bioinformatica Solutions is providing training in all the fields of bioinformatics like vaccine design.si Rna,Bioperl,Biopython,drug designing.The url of the site is

www.thebioinformatica.com]]></description>
      <category>Molecular biology troubleshooting forum</category>
      <guid isPermaLink="true">http://biowww.net/forum/read/5/2480/2480#msg-2480</guid>
      <pubDate>Tue, 25 Mar 2008 01:36:22 -0500</pubDate>
    </item>
    <item>
      <title>[Proteomics and protein biochemistry forum] Re: Semi-dry transfer of high molecular weight proteins</title>
      <link>http://biowww.net/forum/read/18/2477/2479#msg-2479</link>
      <author>mitolab</author>
      <description><![CDATA[There is a old post here: http://biowww.net/forum/read.php?5,8,8#msg-8

Invitrogen has NuPAGE ® Large Protein system that maybe useful. Never tried that. Here is the link: http://www.invitrogen.com/content/sfs/manuals/large_protein_analysis_man.pdf

Good luck.]]></description>
      <category>Proteomics and protein biochemistry forum</category>
      <guid isPermaLink="true">http://biowww.net/forum/read/18/2477/2479#msg-2479</guid>
      <pubDate>Mon, 24 Mar 2008 17:38:32 -0500</pubDate>
    </item>
    <item>
      <title>[Proteomics and protein biochemistry forum] Re: Semi-dry transfer of high molecular weight proteins</title>
      <link>http://biowww.net/forum/read/18/2477/2478#msg-2478</link>
      <author>Mats</author>
      <description><![CDATA[8 hours overnight transfer did not help much. Increase to 12-14 hours at the expense of losing smaller proteins?]]></description>
      <category>Proteomics and protein biochemistry forum</category>
      <guid isPermaLink="true">http://biowww.net/forum/read/18/2477/2478#msg-2478</guid>
      <pubDate>Mon, 24 Mar 2008 08:39:26 -0500</pubDate>
    </item>
    <item>
      <title>[Proteomics and protein biochemistry forum] Semi-dry transfer of high molecular weight proteins</title>
      <link>http://biowww.net/forum/read/18/2477/2477#msg-2477</link>
      <author>Mats</author>
      <description><![CDATA[Hi,

I am having problems transfering anything above 70 KD on to my PVDF membrane using semidry transfer [1 hour transfer at constant 400 mA]. We are using Taubin's buffer generally. So here is what I have done so far:

A) I have added 0.1% SDS to the Taubin recipe [still at 20% methanol]
B) I have reduced the methanol from 20% to 10% and even to 0%
C) I have increased the transfer time to overnight at 50 mA (~4-10 V).
D) I have decided not to equilibrate the membrane in transfer buffer so I keep more of the proteins

Nothing works. Time to quit semidry blotting? There has to be SOMEONE out there that knows the sercret to transfering heavy MW proteins with the semidry method. C'mon folks.............

Cheers!]]></description>
      <category>Proteomics and protein biochemistry forum</category>
      <guid isPermaLink="true">http://biowww.net/forum/read/18/2477/2477#msg-2477</guid>
      <pubDate>Mon, 24 Mar 2008 08:38:03 -0500</pubDate>
    </item>
    <item>
      <title>[Molecular biology troubleshooting forum] Training in bioinformatics</title>
      <link>http://biowww.net/forum/read/5/2476/2476#msg-2476</link>
      <author>shuchi</author>
      <description><![CDATA[We are going to organize a workshop on Training program on Biological sequence analysis from 5-9April 2008,which will include all the basics of bioinformatics.You are kindly requested to give wide publicity to this workshop in your esteemed organization /Institution. The dates and the process for submitting the registration are given in the on the website 
http://www.thebioinformatica.com/trainingProgram.htm

We look forward to your kind co-operation in making this event a grand success. 

Shuchi Smita
Director
The Bioinformatica Solutions]]></description>
      <category>Molecular biology troubleshooting forum</category>
      <guid isPermaLink="true">http://biowww.net/forum/read/5/2476/2476#msg-2476</guid>
      <pubDate>Mon, 24 Mar 2008 06:13:55 -0500</pubDate>
    </item>
    <item>
      <title>[Pharmacology and toxicology forum] Re: ELISA washing</title>
      <link>http://biowww.net/forum/read/29/2474/2475#msg-2475</link>
      <author>mitolab</author>
      <description><![CDATA[The omitting detergent in your washing buffer probably will increase your staining background. If you didn't forget to add it in permeability step it shouldn't be big problem...]]></description>
      <category>Pharmacology and toxicology forum</category>
      <guid isPermaLink="true">http://biowww.net/forum/read/29/2474/2475#msg-2475</guid>
      <pubDate>Fri, 21 Mar 2008 20:33:24 -0500</pubDate>
    </item>
    <item>
      <title>[Pharmacology and toxicology forum] ELISA washing</title>
      <link>http://biowww.net/forum/read/29/2474/2474#msg-2474</link>
      <author>carry</author>
      <description><![CDATA[Hi,

I forgot to add Tween to the wash buffer, and i realized after two washes, i mean i washed the capture Ab without Tween in wash buffer and also after blocking i washed without Tween in wash buffer. 
now my standard and samples are in incubation for 2h,  now i will be washing with 0.05% Tween in PBS.

can you tell me the consequences plz.

Thankyou for your time,

Carry ]]></description>
      <category>Pharmacology and toxicology forum</category>
      <guid isPermaLink="true">http://biowww.net/forum/read/29/2474/2474#msg-2474</guid>
      <pubDate>Tue, 18 Mar 2008 04:46:42 -0500</pubDate>
    </item>
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