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kant0008's Content

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

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#6864 checking for inserts with colony PCR

Posted by kant0008 on 30 August 2004 - 03:43 PM in Molecular Biology

I've had this problem before, with false positives from colony PCR. Do you get a weak ot strong product in your PCR? Sometimes you might have left-over unincorporated DNA in your prep, so that the PCR gives you a positive, even though your vector doesn't have inserts. Do you run a control ligation plate with vector only, no insert? Do you get colonies there? In that case you'll have to modify your cloning procedure.

#6910 checking for inserts with colony PCR

Posted by kant0008 on 01 September 2004 - 03:28 PM in Molecular Biology

are your neds compatible after digesting with RE? Otherwise you shouldn't be getting ANY self-ligated vector.
If they are compatibel- then phosphorylate, and again- no self-anneled vector

#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.

#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.

#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

#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.

#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!

#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?

#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

#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!

#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.

#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.

#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!

#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.

#7375 PCR or RT-PCR?

Posted by kant0008 on 22 September 2004 - 04:29 PM in Molecular Biology

please correct me if I misunderstand your question.
RT-PCR is used for measuring message,you are quite right. The gene shouls almost always be there, unless you are talking special cases, like the Y chromosome, or something, so normal PCR would come up. So go with RT-PCR unless you are working with special cases, or you ar eworking with transfected plasmid DNA- then normal PCR would be ok, as long as you work wuth plamid-specifi, rather than gene specific primers (that could pick up endogenous gene).
Hope that answers your question

#9211 PCR Failure..pls help!

Posted by kant0008 on 06 December 2004 - 09:24 PM in Molecular Biology


You say you get a product of about 80bp- is it a primer dimer? Are your primers complimentary/ self-complimentary? If they are, they could be binding to each other, and so not leaving enough primer available for the reaction. In that case re-design primers.
As far as polymerases go, Hotstar from Qiagen normally works well for me, and people also suggets Omniscript for RT (I myslef haven't tried it).

Also, if you could let us know what you've tried in terms of varying the reaction conditions we could perhaps give more suggestions.

Good luck!

#7103 About R28 cells culture

Posted by kant0008 on 12 September 2004 - 04:56 PM in Cell Biology

how much G418 are you using? They could have some "natural" resistance, depends on what's your dose?
Also, if they have been sitting in culture side by side with transfected lines- unlikely but possible (happened in out lab) cross-contamination can occur. Check for plasmid by PCR?
Also, someone earlier had the same problem with cells that they got from someone else- maybe you don't have all the info?
Hope this helps

#7101 a new molecular biology lab

Posted by kant0008 on 12 September 2004 - 04:44 PM in Molecular Biology


For budget purposes you can get at least some ideas from company websites, such as Promega, Roche, Invitrogen, Qiagen, etc. Otherwise Biocompare.com is not too bad, though some prices are not up to date, but it gives you a range of manufacturers for what you need

#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?

#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!

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