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kant0008's Content

There have been 86 items by kant0008 (Search limited from 15-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

Hi!
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!



#7102 Freezing and thawing of cells

Posted by kant0008 on 12 September 2004 - 04:53 PM in Cell Biology

Hi,
one thing you could possibly try is not to spin your cells straight away, just add the warm medium, and let them sit over a few hours to overnight, then wash out DMSOcontaining medium, and feed with normal medium plus serum (increase serum conc to 10 or even 20% for a little while). Sometimes brittle cells get sheared while centrifuging, so that's why the modified protocol. DMSO is toxic though, so change medium as soon as you see under the microscope that your cells have settled.
If that's the problem you could instead decrease your spin time- even 1min at 1500 should pellet enough cells.
Also, I add medium to my cells, no vice versa, I'm not sure if that would make any difference at all.
As for freezing cells- I do it your way, it seems fine, but your cells could be very sensitive, so could be the problem.
Good luck!



#5926 cloning from PCR

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

Hi,
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?



#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!



#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!



#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!



#7366 PCR failing

Posted by kant0008 on 22 September 2004 - 04:09 PM in Molecular Biology

Hi,
seeing your PCR failed the second time round, even with the small number of samples, it sounds to me like soemthing in your reaction has gone off. If your other PCRs (if you are running any) work, and it's just this one, then your DNA is being degraded. otherwise it could be any of your reagents, dNTPs and Taq being the first ones to check.
Try running out your DNA to check integrity, or spec it for A260.280 ratio. Also change to fresh reagents.
Good luck!



#7434 PCR failing

Posted by kant0008 on 23 September 2004 - 08:48 PM in Molecular Biology

Hi, you said before that your DNA quality is a bot poor, what are your readings or what does it look like? If you say your controls work, then probably the DNA is the problem. Is this genomic DNA? cDNA? Can you do another PCR on it that normally works (liek a housekeeping gene for RT_PCR)?



#7379 PCR failing

Posted by kant0008 on 22 September 2004 - 10:36 PM in Molecular Biology

Hi,
it shouldn't matter when you open your Taq, but rather how it is stored and manipulated (ie, at -20C, always on ice, etc).
Is ther a PCR which always works in your lab you could try?
Or maybe try another vial of Taq?



#5355 correct bands in RT-PCR negative control

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

Hi!
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!



#7306 DNA quantification

Posted by kant0008 on 20 September 2004 - 10:31 PM in Molecular Biology

Re Noel's question,
you are right, gels are only approximate. Spectrophotometry is usually good, as long as your technique is also good, ie good pipetting if you are using a dilution (I normally use 1/50) to measure concentration, clean tubes for blanking, etc. However the spec will give you total nucleotide concentration, which might be prone to error in some cases, eg. if your product is degraded, etc.
what's the experiment you need exact conc for?



#7029 DNA quantification

Posted by kant0008 on 08 September 2004 - 08:54 PM in Molecular Biology

Hi,
do you purify your samples from the gel before sequencing? Why not spec the DNA in that case, you can use a small aliquot of diluted DNA so you don't waste your sample.
As far as markers are concerned: as long as you know what amount of marker you load and you have all the band sizes of the marker from your manufacturer, you can work out the amount of DNA in ng of each band, and by comparing the intensities work out the APPROXIMATE amount of your PCR product. For example, total base pair sum of marker SPP1 is 43787 bases (all bands added together). Then teh top band, which is 8557 bases represents 8557/43787= 0.195 of DNA. If you load 500ng of marker, then your top band will have 0.195X500= 97ng of DNA in it.
Ok, hope this 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.



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

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

Hi!
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.



#7436 Help: try ligation but see nothing from the gels

Posted by kant0008 on 23 September 2004 - 09:10 PM in Molecular Biology

Hi again,
you say you see a difference in fragment sizes cut vs. uncut even with insert? Are you cutting it out of another vector?

OK, about controls.
Ligation positive control: Cut a vector with one enzyme only(so that it is able to reclose), and religate. This should give you a closed plasmid (and colonies if you transform) to make sure ligase is working
Ligation/Transformation negative control: Double digest your vector, purify as normal. Religate vector- if your digest works well then this ligation should Not happen, and you should get zero colonies on transformation.
Transformation positive control: trasform uncut plasmid, should have lots of colonies.



#7373 Help: try ligation but see nothing from the gels

Posted by kant0008 on 22 September 2004 - 04:21 PM in Molecular Biology

Hi,
do you know your plasmid and insert concentrations before you start? You shoudl be able to see at least something, even if you get degradation.
Suggestions:
Don't vortex, pipetting up and down a few times should do the trick.
Don't overheat your mix. 65 should be ok for DNA, but don't leave it too long.
I do my ligations overnight always, at 4C, I don't believe them when they say 1hr is enough :huh:
I agree with labrat, have positive controls so that you know exactly what step is failing. And do check complete digestion too.
Good luck



#7472 Help: try ligation but see nothing from the gels

Posted by kant0008 on 26 September 2004 - 04:12 PM in Molecular Biology

Hi,
just thought I'd mention that to get the cut vector you can go directly by size, as the cut vector will migrate at exactly the molecular size it is, so go by marker, not by intensity



#5927 How to prepare double-stranded oligonucleotides?

Posted by kant0008 on 27 June 2004 - 04:54 PM in Molecular Biology

Hi!
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



#7566 non-viral transfection of MSCs

Posted by kant0008 on 30 September 2004 - 08:52 PM in Cell Biology

Hi,
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...



#7497 non-viral transfection of MSCs

Posted by kant0008 on 27 September 2004 - 09:34 PM in Cell Biology

Hi,
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?



#7281 ligation - stick 3 pieces together

Posted by kant0008 on 19 September 2004 - 08:34 PM in Molecular Biology

Hey postdoc,
100bp is large enough to see, all depends on the concentration of your vector on the gel. But if you get a nice bright band for the vector, then yes, you'd expect at least a very faint band for 100bp fragment



#7368 ligation - stick 3 pieces together

Posted by kant0008 on 22 September 2004 - 04:13 PM in Molecular Biology

hi again,
as far as I understand your cloning is directional, you are using 2 enzymes ? In that case if you do a ligation control with cut plasmid only it should not religate. So then if you run that alongside your annealed product it should run differently if your 100bp fragment is ligated, as ligated plasmid should run higher (normally) thatn cut.
Hope that helps



#7374 ligation - stick 3 pieces together

Posted by kant0008 on 22 September 2004 - 04:23 PM in Molecular Biology

Unless there is somethign reaaly weird going on in your system, no , it shoudln't:)



#7438 ligation - stick 3 pieces together

Posted by kant0008 on 23 September 2004 - 09:23 PM in Molecular Biology

Linear, unligated plasmid will run at the molecular weight, will correspond to marker. Ligated, closed plasmid will run "anywhere", normally somewhere higher than the linear form. So if you mean compare size by base pairs, no it's not possible. If you mean compare size to determine whether the plasmid closed- then yes, it's possible.
The gel percentage- depends on the size of your plasmid, the bigger the plasmid the less concentrated the gel- to have good resolution about the right area. Start with 1%.
Good luck!




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