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kant0008's Content

There have been 86 items by kant0008 (Search limited from 06-August 19)

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#5256 PCR product purification

Posted by kant0008 on 01 April 2004 - 04:48 PM in PCR, RT-PCR and Real-Time PCR

I have never tried Zymogen kits, but Qiaquick works well normally. A few possible problem areas:
1) How big is your PCR product? Has to fall within the limits specified in the kit
2) Qiaquick is the gel kit, right? So you actually have to cut out your product. How intense was your band of interest? Maybe your PCR wasn't particularly good
3) Check the pH of your elution buffer, or (especially!) water, DNA will not be eluted if pH is too low, has to be slightly basic. (MQ water is not nevessarily neutral)
4) How old is your isopropanol? It's hydrophilic, so if it gets opened too often, it will be "diluted", so your concentrations will change. Get a new batch
Hope this helps!

#5355 correct bands in RT-PCR negative control

Posted by kant0008 on 14 April 2004 - 11:56 PM in Molecular Biology

If your contamination occurs only in 4 out of 240 reactions this is a rare event, you must be getting contaminants in some of your buffers or other reagents, but the concentration of contaminating DNA must be low, so that when you aliquot your samples you don't get enough template in many reactions. Get fresh buffers/ aliquot, and discard as soon as contamination apperas.
As for your actin problem- it is not actually as good a housekeeping gene as some people believe. I don't know what your system is, but your treatment may actually influence actin, try a different control, such as GAPDH, PBDG or even 18S rRNA.
Good luck!

#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.

#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.

#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!

#5725 vector pFPV25.1

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

Do you have the sequence of this vector?

#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!

#5926 cloning from PCR

Posted by kant0008 on 27 June 2004 - 04:46 PM in Molecular Biology

I have very strong doubts you'd be able to see such small fragments no matter what you do. How far into your PCR products are your restriction sites? Enzymes normally need at least 6 bases distnace from recognition site to be present to cut. Also, do you have a transformation control (same plasmid but undigested, no insert) to make sure your cells are still competent?

#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

#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.

#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.

#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?

#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.

#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

#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.

#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!

#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!

#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?

#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!

#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.

#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.

#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.

#6341 Cloning long fragments

Posted by kant0008 on 03 August 2004 - 05:08 PM in Molecular Biology

some possible steps you could check:
1)is your ligation working?
I would test ligase on some other DNA, eg. you could purify BOTH your vector and small insert that you normally discard (how big is it? Qiagen kits go down to about 70bp) and then religate. Then either run on gel to visualise (run with closed vector, and cut vector as controls to copmare against), or transform into bugs and check for colonies.
Also you can run a sample of your actual ligation reaction on gel, and if you get good ligated plasmid then transform the rest.
I do my ligations overnight at 4C, or even over a weekend, no harm in going for a longer time.
2)As Sprag said, is your transformation working?
I always do a control, eg. your initial vector, that should give you plenty of colonies. Vector without insert is a good negative control, but going by your enzymes the vector should not reclose on itself, plus you'd get colonies that way, so I don't think that's the problem.
3) Your dialysis step- I don't bother, perhaps you lose DNA there somehow? If you do it, check DNA concentration afterwards
4) Have you tried other ratios of vector to insert? Could help
5) Do you have bacterial control, ie is your amp concentration ok?
Good luck!

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