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kant0008's Content

There have been 86 items by kant0008 (Search limited from 14-August 19)

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#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?

#6271 western blotting, (GFP is there

Posted by kant0008 on 29 July 2004 - 07:52 PM in Protein and Proteomics

Could it be out of frame?
Do you have a positive control for the antibody?

#6272 Western Blotting Help!!!!

Posted by kant0008 on 29 July 2004 - 07:57 PM in Protein and Proteomics

Hi again,
I would try a different housekeeping gene, there are various examples in the literature, we have previosuly used PBGD. Actin might not be too good a control.
if you want to test for shock response try an RT-PCR for one of the heat shock proteins, you might observe a large increase in expression - I have never done this, so don't much about it. Again literature might be helpful. The reason I though of a shock response inthe first place is because you say you observe decreased cell growth rate and viability. Hope thos helps and didn't confuse you even further.

#6262 Western Blotting Help!!!!

Posted by kant0008 on 28 July 2004 - 08:25 PM in Protein and Proteomics

It has been noted, especially recently, that b actin is not really that great a control, its levels change during various cellular events. So no surprises there. You say you tried GAPDH, which is another housekeeping gene. Another one is PBGD. Are you sure you don't have a global shut down of protein expression, like a shock response?

#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

#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!

#5725 vector pFPV25.1

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

Do you have the sequence of this vector?

#5369 use Pen./Strep. in the media for trasnfection

Posted by kant0008 on 15 April 2004 - 07:13 PM in Molecular Biology

I tried Calc Phos, but it didn't seem to be particularly good. A similar methodology, but using Dextran sulfate normally works better, but there are problems with toxicity, so you got to watch it. Lipofectamine seems quite good, but you might still try smth different, like Fugene6 or DOTAP, there are tons of different reagents on the market now.

#5356 use Pen./Strep. in the media for trasnfection

Posted by kant0008 on 15 April 2004 - 12:03 AM in Molecular Biology

Hi guys!
I add pen/strep to all my cultures, including transfections. You're better off in most cases with a lower transfection efficiency than no cells at all due to contamination. Some transfection reagents/methods work better with some cells and not others, so I suggest use pen/strep but maybe look at other transfection methods.

#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.

#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.

#5925 solubility of dna

Posted by kant0008 on 27 June 2004 - 04:35 PM in Molecular Biology

I agree with hula, it could be something other than dna that you've isolated. Otherwise could be a number of things:
1) Did you have an ethanol wash as the last step of your protocol? If the ethanol wasn't dried off properly your nucleic acid will not dissolve. I normally dry my samples with a pipete tip (capillary action, upside down) and then leave them for a while with lids open (cover with tissue or something) on bench or ona heating block (not too hot). With the samples you alredy have you can do an ethanol precipitation and redissolve
2) Your dna might be very concentrated and so reached the extent that it'll dissolve, what's the concentration? Try adding more buffer.
3) Leave your dna at 4C overnight to allow to dissolve, genomic dna takes a while to dissolve
Hope this helps!

#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.

#6713 Selection of clones during transfection

Posted by kant0008 on 22 August 2004 - 10:04 PM in Cell Biology

can you find out at what concentration cells will die? Construct a kill curve? It takes about 2weeks.
Otherwise 0.4-0.8mg/ml seems to be the range for most cell lines I use

#6845 Selection of clones during transfection

Posted by kant0008 on 29 August 2004 - 11:08 PM in Cell Biology

Hello again,
if you say your cells are sensitive then you can start with a low concentration of antibiotic. Theoretically for a sensitive cell line a small amount of it should still be enough to maintain a selective pressure. You can always run a control non-transfected well, to make sure you are not getting random mutants. If you have the option I'd still try a kill curve, it might actually save you time and cells in the long run.
Good luck!

#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!

#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

#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.

#6542 Restriction Digest

Posted by kant0008 on 12 August 2004 - 03:36 PM in Molecular Biology

this is just some stuff I founf in NEB catalog, so aaI apologise if you've already thought of it all:
1)What's your reaction pH- apparently at pH7.0 the digestion needs to be at 25C, not 37C
2)Some sites apparently are cut really slowly, so go with the longer incubation time
3)Is your DNA methylated by any chance- this enzyme is blocked by CpG methylation

I'd check your enzyme on some other DNA.

#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

#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

#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.

#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!

#6080 Problems in bisulfite sequencing reproducibility

Posted by kant0008 on 12 July 2004 - 10:24 PM in DNA Methylation and Epigenetics

is your sequence being completely converted by bisulfite? As in are you getting Cs only at CpGs or somewhere else too? Make sure your bisulfite is not off, as well as other reagents, and obviously use the same protocol.
If you are talking about the same DNA sample that's the only thing I can think of. I agree with pcrman in that cloning and then sequencing clones might give you a better idea of what methylation frequency of your sites is-sites can be methylated or not within the same cell population.
Or did you extract a second lot of DNA and then bisulfite modified that? Methylation can occur spontaneously over time on cell culture.

#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?

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