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kant0008's Content

There have been 86 items by kant0008 (Search limited from 10-April 19)



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#6542 Restriction Digest

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

Hi,
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.



#6516 Cloning long fragments

Posted by kant0008 on 11 August 2004 - 06:20 PM in Molecular Biology

Hi!
No wonder you are frustrated!;) This sounds like a pain!
Hm, about your ligation on gel: what did you see except for the smear? You should see a lot of ligated plasmid (which runs somewhere at the top of the gel, higher than the plasmid size), you may also see some unligated plasmid at the plasmid size. As long as you see the ligated plasmid it should be ok.
Did you try your ligase on some unrelated DNA?
Running out of ideas:(
Good luck!



#6515 Methylation and restriction digestion problem

Posted by kant0008 on 11 August 2004 - 05:50 PM in Molecular Biology

Hi,
are you completely positive your fragment was incorporated? Sometimes PCR can come up with positives even if there are just a few small fragments around in your DNA prep (I realise it shouldn't happen with pure DNA, but sometimes it does).
I would try to cut your construct with an anzyme or two that are only founf in the insert, say somewhere in the middle- see if it cuts.



#6490 Annealing temperature for PCR

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

Hi,
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



#6426 Cloning long fragments

Posted by kant0008 on 08 August 2004 - 05:22 PM in Molecular Biology

Hi again,
sounds like you ar ehaving "fun":wacko:
Ok, transfrormation. I'd use your initial vector- don't cut and ligate, just the original plasmid, as the transformation control. If it doesn' give you good number of colonies you'll know that it's not the ligation and there is something wrong with the transformation.
If it is the transformation that doesn't work you can try making fresh competent cells, it may be the problem (how do you make them? I can give you an easy protocol). Double check the ampicillin conc just as an extra precaution.
I don't think water could harm your reaction in any way, it's only water.
Not sure about broth, I've not used it myself, but theoretically there shouldn't be anything in there that inhibits bacterial growth.
And what do you use TAE for? Is it in the Qiagen prep step? I use water, it works ok, just leave your tubes 5mins before spinning for the last time.
Good luck!



#6425 Bands are like a "w"

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

Hi,
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.



#6383 vector with no sp1 in promoter

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

Hi,
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!



#6378 RT product check

Posted by kant0008 on 05 August 2004 - 03:33 PM in Molecular Biology

Hi,
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



#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?



#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

Hi,
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.



#6341 Cloning long fragments

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

Hi,
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!



#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.



#6299 a question about mutagenesis

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

Hi,
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.



#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.



#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?



#6270 a question about mutagenesis

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

Hi!
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!



#6262 Western Blotting Help!!!!

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

Hi!
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?



#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!



#6258 RT-PCR, why two PCR steps?

Posted by kant0008 on 28 July 2004 - 05:02 PM in Molecular Biology

Hi!
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!



#6215 passage number

Posted by kant0008 on 25 July 2004 - 08:23 PM in Cell Biology

Hi,
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.



#6210 PCR is6110 Tuberculosis

Posted by kant0008 on 25 July 2004 - 03:48 PM in Molecular Biology

Hi!
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



#6080 Problems in bisulfite sequencing reproducibility

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

Hi,
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.



#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?



#5974 PCR Cloning

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

Hi!
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.




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