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kant0008's Content

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

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#6383 vector with no sp1 in promoter

Posted by kant0008 on 05 August 2004 - 05:45 PM in Molecular Biology

I'm trying to find a plasmid vector with some sort of a detectable product that has no sp1 sites in its promoter, as I'm trying to monitor the effect of sp1 on my gene of interest and want to find a plasmid I can use as a transfection efficiency control.
Thanks for any suggestions!

#7127 ELISA normalisation

Posted by kant0008 on 13 September 2004 - 11:12 PM in Protein and Proteomics

Hey all,
just wondering, when you do ELISA do you dilute your samples to a uniform total protein concentration and then do the assay, or is it ok to use eg. a constant volume of sample and then work out the concentration of protein of interest later, based on a Bradford?

#6425 Bands are like a "w"

Posted by kant0008 on 08 August 2004 - 05:09 PM in Molecular Biology

normally strange band shape is to do with the gel, for example agarose not dissolving properly before you por so there are clumps, or setting slightly before you pour- again causing small clumps that would cause the sample to run unevenly. Try decreasing the agarose conc and make sure it all dissolves properly. It could theoretically be a problem with your power supply, but then you'd get a strange pattern all over your gel, not the same for all bands.

#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

#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.

#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.

#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.

#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.

#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?

#7028 Western Blots

Posted by kant0008 on 08 September 2004 - 08:42 PM in Protein and Proteomics

Incubation with milk in theory should give you a cleaner western with less background noise

#6315 his and myc tags

Posted by kant0008 on 02 August 2004 - 06:20 PM in Protein and Proteomics

A his-tag is a technique used in biochemistry to purify proteins. The protein to purify is expressed as a fusion protein, carrrying a series of histidine residues at either the amino or the carboxyl end. The name-giving histidine residues will stick to a nickel surface, and can be eluted from there via imidazol. His-tagged proteins can also be identified on a Western blot via -his-antibodies.
Myc tags are similar.

#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!

#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.

#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!

#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?

#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..

#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?

#5725 vector pFPV25.1

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

Do you have the sequence of this vector?

#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.

#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:)

#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!

#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.

#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

#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

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