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kant0008's Content

There have been 86 items by kant0008 (Search limited from 19-September 19)

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#6843 Stable transformants

Posted by kant0008 on 29 August 2004 - 08:27 PM in Cell Biology

resistant cells will continue growing and dividing in the presence of antibiotic, and you will get more and more dead cells with time in sensitive cell lines- I do my curves over 14 days. If you are trying to pick a concentration to select stable clones with- I normally go for the one that gives you dramatic cell death by about day 5, and no live cells at all by day 14.
As far as Shilpi's question goes: I'm not familiar with your particular cell line, but cell lines differ in their susceptibility to geneticin, and for some of my cells I have gone up to 1.2mg/ml. Change media as needed, as you would in normal tissue culture.

#6715 EMSA problem

Posted by kant0008 on 22 August 2004 - 10:11 PM in Protein and Proteomics

try running your gel at 4C if you have the option to do it, your protein-DNA complex might be labile at RT.
Also if you are getting a smear (nonspecific binding?) try decreasing your protein amount, try 1ug or even less. Also with a smear sometimes it helps to add extra non-specific competitor, like polydIdC-polydIdC, try 0.2ug for every 5ug protein. If you get no bands at all, then don't add competitor at all, see what happens.
Good luck!

#6362 wierd plasmids: maxi-prep never work. ###HELP####

Posted by kant0008 on 04 August 2004 - 04:36 PM in Molecular Biology

Do your maxipreps have plasmid size specifications? Could they be different to those for mini-preps?

#7101 a new molecular biology lab

Posted by kant0008 on 12 September 2004 - 04:44 PM in Molecular Biology


For budget purposes you can get at least some ideas from company websites, such as Promega, Roche, Invitrogen, Qiagen, etc. Otherwise Biocompare.com is not too bad, though some prices are not up to date, but it gives you a range of manufacturers for what you need

#7103 About R28 cells culture

Posted by kant0008 on 12 September 2004 - 04:56 PM in Cell Biology

how much G418 are you using? They could have some "natural" resistance, depends on what's your dose?
Also, if they have been sitting in culture side by side with transfected lines- unlikely but possible (happened in out lab) cross-contamination can occur. Check for plasmid by PCR?
Also, someone earlier had the same problem with cells that they got from someone else- maybe you don't have all the info?
Hope this helps

#9211 PCR Failure..pls help!

Posted by kant0008 on 06 December 2004 - 09:24 PM in Molecular Biology


You say you get a product of about 80bp- is it a primer dimer? Are your primers complimentary/ self-complimentary? If they are, they could be binding to each other, and so not leaving enough primer available for the reaction. In that case re-design primers.
As far as polymerases go, Hotstar from Qiagen normally works well for me, and people also suggets Omniscript for RT (I myslef haven't tried it).

Also, if you could let us know what you've tried in terms of varying the reaction conditions we could perhaps give more suggestions.

Good luck!

#7375 PCR or RT-PCR?

Posted by kant0008 on 22 September 2004 - 04:29 PM in Molecular Biology

please correct me if I misunderstand your question.
RT-PCR is used for measuring message,you are quite right. The gene shouls almost always be there, unless you are talking special cases, like the Y chromosome, or something, so normal PCR would come up. So go with RT-PCR unless you are working with special cases, or you ar eworking with transfected plasmid DNA- then normal PCR would be ok, as long as you work wuth plamid-specifi, rather than gene specific primers (that could pick up endogenous gene).
Hope that answers your question

#6259 Methods for mechanical disruption of cell

Posted by kant0008 on 28 July 2004 - 05:09 PM in Cell Biology

Hi all!
This is not a reply, but I thought I'd translate the question so maybe someone will be able to help Sebela:)
Question goes something like (my french is not 100%):
"does anyone have any efficient methods for mechanical disruption of cells (lymphocytes)? I'm having problems in getting complete lysis of my cells so I'd like to try 2 or 3 methods consecutively"

Bon chance Sebela!

#6669 EMSA problem

Posted by kant0008 on 19 August 2004 - 03:37 PM in Protein and Proteomics

could you specify your conditions? Do you use a kit? There are a few things you could try, like changing protein amount, running at 4C and no loading buffer, making sure your probe is ds, etc. But we need to know what methods you are currently using

#5927 How to prepare double-stranded oligonucleotides?

Posted by kant0008 on 27 June 2004 - 04:54 PM in Molecular Biology

My method's pretty similar. I add equimolar amounts of both oligos, but if you dissolve oligos in water, you have to then add STE buffer (I find as high as 90% STE total volume is good). Then heat at 95 for about 15 mins, and allow to cool slowly to room temperature- either use a heating block, and take the metal block off onto the bench, or use a pcr cycler (gradually cool). As far as purification, run on gel and purify. In theory shouldn't matter, as ssDNA will not be incorporated into anything you're working with, such as plasmids

#7754 proliferation assay

Posted by kant0008 on 11 October 2004 - 09:31 PM in Cell Biology

What's your Northern for? What's your probe? Maybe it's some gene that is involved in proliferation? In that case it would be just another way to double-check your results. I guess.
As for your media, if your cells are happy you're probably ok. But if you feel uneasy wash out your wells as normal, add fresh mediaum, drug and whatever else is in your asssay- this of course would mean you are using up more reagents. But then aagain, how stable are your chemicals in tissue culture conditions- if not very stable you'll have a better chance that your cells have been subjevted to the effect of your chemicals for the entire week.

#7496 H295R Cell transfections

Posted by kant0008 on 27 September 2004 - 09:31 PM in Cell Biology

I don't know anything about the cells you are using but with my cells which were difficult to transfect Lipofectamine helped a lot (from Invotrogen). You can also try the home-made Dextran sulfate method. It is ok as far as transfection goes, but is very toxic, so might be suboptimal.

#8408 Double Digest problems

Posted by kant0008 on 03 November 2004 - 10:21 PM in Molecular Biology

the fact that your undigested plasmid is lower than double digest wouldn't worry me, uncut vectors run anywhere almost:) The fact that your single-digested vector runs higher though suggests to me that it's not a complete digest and your plasmid might be just getting nicked- single strand cut, which means that it'll get unwound and so tangle up wothin the gel. Make sure you have a godd site within the vector and that your digest has enough enzyme, appropriate buffer and continues for enough time.
Good luck!

#8818 Cloning pCAEGFP into XL1 E. coli

Posted by kant0008 on 22 November 2004 - 03:38 PM in Molecular Biology

Some steps to check would be:
1) The efficiesncy of your restriction digest: are you digesting your insert out of another vector, or is it PCR? What about the vector itsef? Basically, are you sure you are getting the fully digested product?
2) Run a sample of ligated product on gel, check that you are actually getting ligation happening
3) test your bugs with another vector (+ve control) for ability to get transformed
4) check the concenration of kana in your plates, with a different bug say.

Depending on these results you could figure out your problem. Good luck!

#9311 Cell contamination, needs the help urgently

Posted by kant0008 on 09 December 2004 - 09:39 PM in Cell Biology

sorry to tell you this, but contamination (bacterial, fungal, micoplasama) is VERY hard to remove, if not impossible! you can try harsh antibiotics, nut it might not work, or your cells will die..

#5725 vector pFPV25.1

Posted by kant0008 on 01 June 2004 - 09:27 PM in Microbiology

Do you have the sequence of this vector?

#5959 Problem with plasmid linearization

Posted by kant0008 on 29 June 2004 - 09:59 PM in Molecular Biology

Hi there!
A linearised plasmid should run at the size corresponding to its actual bp length. Uncut or nicked plasmids may run at various sizes, but should be consistent from time to time. Your 2 bands could be uncut and nicked DNA, ie some partial digestion maybe?

#5960 Stable transfecion of BE2(M17)

Posted by kant0008 on 29 June 2004 - 10:04 PM in Cell Biology

Could your antibiotic be old? G418 has to be stored in the freezer and in the dark.
If it's ok, then try waiting another 2-3 days or increase concentration further(?).
Also, somewhat unlikely but possible, could your cells be contaminated by a G418 plasmid of some sort? It could happen if you have done many transfections before.

#6378 RT product check

Posted by kant0008 on 05 August 2004 - 03:33 PM in Molecular Biology

I agree with pcrman about your RNA: does it give you the correct pattern, good clean sample, no smearing, the top band being about 2X as intense as bottom?
Also true about pcr- does your positive control come up? Did this PCR ever work?
Did you run an RT control PCR on these samples? For example bActin or GAPDH, or another housekeeping gene. These should give you a product, so you know your RT worked

#6215 passage number

Posted by kant0008 on 25 July 2004 - 08:23 PM in Cell Biology

basically every time you trypsinise your cells (they are adherent?) you increase the passage number by one. That means if you split them even at 10times the suggested amount it still goes up by one. Also when freezing you would have trypsinised them before storage, therefore even though the revival itself does not add a passage to your count, the number would be 24.

#5422 Cell Cycle/DNA/RNA Unanswered Questions

Posted by kant0008 on 21 April 2004 - 10:06 PM in Cell Biology

I do apologise, but I believe some of the answers above are incorrect.
Here's my version, choose whichever you like:
2= (not 100%sure, but A sounds closer to the truth)
8= A (no doubt about this one!)
Good luck!

#6711 Importance of volume of Flascon tubes

Posted by kant0008 on 22 August 2004 - 09:52 PM in Molecular Biology

the area that you are growing your bacteria in makes a difference to the yield, I would definitely go with the 50ml tubes. Obviously this is going to increase your amount only, not give you growth if you have none:)

#6034 mycoplasma and fungus

Posted by kant0008 on 07 July 2004 - 05:11 PM in Cell Biology

Hi all!
I'm trying to find out about fungul and mycoplasma contamination. The other day I gave some of my cells to a collegue who is studying fungus, as a negative control, yet my cells came up positive in his pcr. he seems quite certain that it's a not a false positive. My cells seem fine, and I can see no usual fungal "characteristics"- long filaments or lawns. However there are a few floating "objects" in the flasks- they are not of a regular shape, can form clumps, about a quarter of the size of my cells (so too big for bacteria). I can't figure out if they multiply at all, there seem to be a few at all times, with a small increase in number over time- but what I am not sure about is if they multiply or if some of my cells are dying and that's just debris. Can it be mycoplasma? Woudl I be able to see it under a microscope? Is there any way to treat this infection?
At the moment I'm adding 4 different antibiotics to these cultures, as they are transfectants and I'm keeping two plasmids, so the usual Pen/strep mixture, plus neomycin and hygromycin (that's why I was surprised to get an infection, if that's what it is, and also think that maybe my cells dye faster with all tese antibiotics).
Any comments please?

#6210 PCR is6110 Tuberculosis

Posted by kant0008 on 25 July 2004 - 03:48 PM in Molecular Biology

I'm not familiar with MTB PCR, however from general PCR I'd say that it could be
1) contamination as postdoc said. Do your negative controls come up with any bands? If so throw out all solutions, start again.
2) Seeing you are getting some correct fragments, but not all, could you have polymorphisms of your gene? Therefore giving you different sizes? If these facilities are avaiable try sequencing your 400bp product and examine the sequence.
3) As I said I'm not sure what MTB PCR is, so disregard this point if I'm off track- is it a DNA, RNA PCR or both? You could possibly be amplifying a larger fragment off DNA.
Hope some of the above helps

#6258 RT-PCR, why two PCR steps?

Posted by kant0008 on 28 July 2004 - 05:02 PM in Molecular Biology

I'm not sure if I understood your question right: is it that there is a reverse transcription step and then 2 PCRs, or as pcrman suggests the whole thing is together in one step, or a single RT and single PCR?
If it is RT then 2 PCRs it's called nested PCR (the second lot of primers is within the first lot, giving smaller product). It is used for rare products (withcraft, no product could appear in first pcr, but will turn up in second) or when you want to be a bit more specific with your product (smaller chances of amplifying irrelevant sequences), or specific applications, such as mutagenesis.
I would try a single PCR for your siRNA work, and if it works stick with it, if not then try a 2step reaction
Good luck!

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