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kant0008's Content

There have been 86 items by kant0008 (Search limited from 29-February 20)

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#12782 MMP assay

Posted by kant0008 on 22 March 2005 - 09:40 PM in Cell Biology

You could make cell extracts- cytoplasmic for eg, without adding protease inhibitors, as long as you work quickly and in cold conditions it shoul dbe ok.

#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..

#9253 semi-dry transfer

Posted by kant0008 on 07 December 2004 - 08:58 PM in Protein and Proteomics

if you also swapped over the anode/cathode wires (plugged into the wrong side, or up in the powerpoint) your blot would work as normal. That's the only thing I can think of at the moment.

#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!

#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!

#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!

#7992 plasmid digest

Posted by kant0008 on 18 October 2004 - 11:07 PM in Molecular Biology

Are you sure that your plasmid is what yopu think it is?
How many sites for EcoRV is in it?

#7991 RNA Isolation

Posted by kant0008 on 18 October 2004 - 11:04 PM in Molecular Biology

Why not give it a go with a couple of samples? Also depends on what exactly you are planning to do with it, some techniques are more sensitive than others to contaminants and degradation.

#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.

#7753 Help with stable cell lines

Posted by kant0008 on 11 October 2004 - 09:25 PM in Cell Biology

as to your amount of selection antibiotic, it doesn't really matter what you use as long as you have healthy cells and enough of a selection pressure at all times. I personally like hitting cells with a high dose, and when only a few are left I decrease the antibiotic.
You say that [QUOTE]Many cells died but there are still so many cells so I cannot pick isolated colonies with a Gilson,

Are you sure that you have stably transfected cells, are you using enough G418? Normally only a few cells survive, so that you should be able to pick colonies.

#7682 Agarose gel issue

Posted by kant0008 on 06 October 2004 - 11:27 PM in Molecular Biology

try ligating overnight at 4C, or even over a couple of nights.
Also, ligase buffer should have ATP in it, you sure you're not overdoing it?
Also use no more than 1/10th of total volume of ligase, it's storage buffer may interfere (eg. only up to 2ul for 20ul reaction)
Good luck

#7665 Digestion time and enzyme

Posted by kant0008 on 05 October 2004 - 09:24 PM in Molecular Biology

as for time- how long is too long? Some enzymes have "star" activity, they start cutting non-specifically if at too high concentration or if left too long, I'm not sure if Bam HI has it though. I don't think there are any specific requirements for cutting PCR products.
To get rid of your enzyme you can ethanol precipitate your mixture.

#7639 digestion & purification problem

Posted by kant0008 on 04 October 2004 - 10:55 PM in Molecular Biology

yeah, i agree with mario, you can ethanol precipitate, but I find that you tend to lose more DNA than with gel kits. Also, less than 60% recovery is not too flash for a kit, which one have you used? I use Qiagen gel kit, and it's ok, although I must say that the smaller fragments (abut 100bp) are lost at a much higher rate- but for you that should be ok. Also sometimes you need to optimise the protocol of the kits just a little bit, eg incubate with elution buffer for longer.
Good luck

#7566 non-viral transfection of MSCs

Posted by kant0008 on 30 September 2004 - 08:52 PM in Cell Biology

yes that is what I meant. OK, so have you tried to transfect these cells before, with someother vector? Just to make sure that it's a problem with transfection...

#7565 Problems with TOPO cloning

Posted by kant0008 on 30 September 2004 - 08:42 PM in Molecular Biology

the fact that you got 2 PCR products makes me think that your primers can bind twice along the template, one inside the other. That means that if you provide the 1.4 kb sequence as your template you theoretically should get 2 bands again. But the smaller one will be easier to amplify, so you might get bias. Try to do a restriction digest on your plasmid from transformed cells, with an anzyme that you know the sites for and see what size fragments you get. Good luck!

#7541 5-Aza-2'-deoxycytidine

Posted by kant0008 on 29 September 2004 - 06:00 PM in DNA Methylation and Epigenetics

as far as I know 5-Aza-C is quite toxic to cells and they can't survive very long with it. It depends on the concentration of course. But it's not a "nice" chemical

#7497 non-viral transfection of MSCs

Posted by kant0008 on 27 September 2004 - 09:34 PM in Cell Biology

could it be that your vector is not activated when transfected? Are you sure that it works, did you see expression and glowing with it somewhere else?

#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.

#7473 question about stable transfection

Posted by kant0008 on 26 September 2004 - 04:23 PM in Cell Biology

it depends on your cell growth also, but G418 is stable for about 4 days, so if your cells don't become overconfluent or start looking sick and like they need a "feed" once in 4 days should be ok

#7472 Help: try ligation but see nothing from the gels

Posted by kant0008 on 26 September 2004 - 04:12 PM in Molecular Biology

just thought I'd mention that to get the cut vector you can go directly by size, as the cut vector will migrate at exactly the molecular size it is, so go by marker, not by intensity

#7438 ligation - stick 3 pieces together

Posted by kant0008 on 23 September 2004 - 09:23 PM in Molecular Biology

Linear, unligated plasmid will run at the molecular weight, will correspond to marker. Ligated, closed plasmid will run "anywhere", normally somewhere higher than the linear form. So if you mean compare size by base pairs, no it's not possible. If you mean compare size to determine whether the plasmid closed- then yes, it's possible.
The gel percentage- depends on the size of your plasmid, the bigger the plasmid the less concentrated the gel- to have good resolution about the right area. Start with 1%.
Good luck!

#7437 Proteinase digestion of tissue for DNA extraction

Posted by kant0008 on 23 September 2004 - 09:12 PM in Molecular Biology

You can also freeze your tissue pieces in liquid nitrogen and then grind to powder before digesting

#7436 Help: try ligation but see nothing from the gels

Posted by kant0008 on 23 September 2004 - 09:10 PM in Molecular Biology

Hi again,
you say you see a difference in fragment sizes cut vs. uncut even with insert? Are you cutting it out of another vector?

OK, about controls.
Ligation positive control: Cut a vector with one enzyme only(so that it is able to reclose), and religate. This should give you a closed plasmid (and colonies if you transform) to make sure ligase is working
Ligation/Transformation negative control: Double digest your vector, purify as normal. Religate vector- if your digest works well then this ligation should Not happen, and you should get zero colonies on transformation.
Transformation positive control: trasform uncut plasmid, should have lots of colonies.

#7434 PCR failing

Posted by kant0008 on 23 September 2004 - 08:48 PM in Molecular Biology

Hi, you said before that your DNA quality is a bot poor, what are your readings or what does it look like? If you say your controls work, then probably the DNA is the problem. Is this genomic DNA? cDNA? Can you do another PCR on it that normally works (liek a housekeeping gene for RT_PCR)?

#7379 PCR failing

Posted by kant0008 on 22 September 2004 - 10:36 PM in Molecular Biology

it shouldn't matter when you open your Taq, but rather how it is stored and manipulated (ie, at -20C, always on ice, etc).
Is ther a PCR which always works in your lab you could try?
Or maybe try another vial of Taq?

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