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kant0008's Content

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

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#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?

#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...

#7198 Problem with PCR on difficult template

Posted by kant0008 on 15 September 2004 - 08:10 PM in Molecular Biology

from personal experience AmpliTag is not a very good enzyme. At least in my hands it wasn't.
If I have problems with PCR using standard Taq (I use the one from Promega), then I go to Qiagen's HotStar. It works on most difficult templates. Also they provide something called Qsolution (some buffer I guess), which also can make a difference.
If you want to try with your Taq, add a little formamide or glycerol (work similarly to DMSO) they help with amplifying GCrich templates.
Good luck

#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

#7307 3' end of forward primer mismatch

Posted by kant0008 on 20 September 2004 - 10:34 PM in Molecular Biology

5 bases at the end of a primer sounds like a lot, I wouldn't think it would work. Depends how long it is as well. Do you know whether it worked well for your collegue?

#6712 If the primer is dimer,it's ok?

Posted by kant0008 on 22 August 2004 - 09:56 PM in Molecular Biology

I guess it depends on how much of the sequence dimerises. If it's a lot then you may not have any free primer for the PCR reaction to proceed, so you won't get a product.

#6346 A question about adenovirus infection 293 cells

Posted by kant0008 on 03 August 2004 - 10:43 PM in Molecular Biology

Hi again,
about your questions:
yes, 2ug vector works well, with 5ul Lipo.
I normally plate about 1X10.5- 5X10.5 cells per well of a 6well plate, but it's different for different cells, just go with the number that will give you 90% confluence after 24hrs. And yes, I do count.
Again I personally don't bother changing the medium after transfection, but my cells are quite robust and if you are having problems with toxicity I'd change the medium, 6hours is probably best.
Hope this helps

#6342 A question about adenovirus infection 293 cells

Posted by kant0008 on 03 August 2004 - 05:14 PM in Molecular Biology

I probably misunderstood you, but are your cells in serum-free media for 6 hours? I actually keep my cells with serum in media at all times, serum free media is only required for incubation of Lipofectamine and plasmids. So I don't change media before I transfect, just add Lipo with plasmid in serum-free media to ready wells.
Also you could decrease the concentration of Lipo, I find 5ul per well works fine.

#6270 a question about mutagenesis

Posted by kant0008 on 29 July 2004 - 07:42 PM in Molecular Biology

I'm not sure how the stratagene pcr is supposed to work, since you are adding complementary primers to your pcr, and in normal pcr that never gives product. I'm sure I'm missing something. Maybe someone else knows how it works? Would be interesting to read an explanation.

But I use a different pcr-based method, using pfu turbo taq, which is quite easy and involves 4 primers: 2 of your complementary primers carrying the mutation plus 2 plasmid-specific primers somewhere away from mutation. Basically you run pcrs with 3'primer + one oligo (corrects strand) and 5'oligo+ the other oligo. Purify products of those reactions and use them to run a third pcr, with outlying primers only, then clone back into your original plasmid sequence (need to have suitable restriction sites).
Good luck with whichever method you pick!

#6299 a question about mutagenesis

Posted by kant0008 on 01 August 2004 - 03:18 PM in Molecular Biology

if you do go ahead with this method (and it seems other people have used Statagene succesfully so you may want to stick with them), the plasmid-specifi primers are designed the same way as any pcr primer, eg run primer 3 to give you possible sequences, check them with a pcr prediction program such as amplify, check tms etc, check with blast that they are ok and won't amplify other genes. The only thing is make sure your pcr product in the end is nott too big, or will be difficult to amplify.

#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!

#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

#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

#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.

#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?

#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.

#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!

#5974 PCR Cloning

Posted by kant0008 on 30 June 2004 - 08:17 PM in Molecular Biology

It sounds to me like your primers are not the problem if you say you are getting the right pcr product. However, if you are digesting your pcr product for ligation, the emzymes may not cut- sounds like you only have 3 bases (ATT) at the end, this is too short, enzymes (with some exeptions) require at least 6 bases to from recognition site to cut properly.
Also, the reason you are getting colonies could be
1) Your plasmid is self ligating- this is only possible if your 2 enzymes generate compatible ends, check this eg. at New England Biolabs site
2) You have uncut plasmid in the ligation mix- do you purify your cut plasmid before ligation?

About methylation blocking restriction: again, check NEB site, it will say if Not I is affected. Is your DNA methylated? Normally DNA propagated in commonly used bug strains is not methylated- check your strain.

#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

#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.

#6490 Annealing temperature for PCR

Posted by kant0008 on 10 August 2004 - 09:36 PM in Molecular Biology

normally I'd subtract from the Tm of smaller primer. If you are worried about the specificity of your reaction you can do a test run with a few different annelaing temperatures

#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

#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

#6865 my bands spread as a line trough the gel

Posted by kant0008 on 30 August 2004 - 08:09 PM in Protein and Proteomics

yes, make sure there's no overloading and so leakage from one well into the next. Also make sure that your spacers and combs are the same thickness- if your combs are too thin you will get gel everywhere, and might be loading your samples into the gel, embedding everywhere, rather than neatly into wells.

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