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this may be a silly question, but i was wondering whether you need to linearise your plasmids before transfecting them into eukaryotic cells for stable transfection. i've heard arguements for and against. what do you guys do?
I am after a protein quantitation method which is ok to use when there is detergents in your solubilisation/lysis buffer. I would normally use the Biorad protein assay (Lowry assay), but I am getting massive errors (always increased estimates) of my protein concentrations. I have been using trizol to extract the protein so I assume it is due to a carry-over of phenol (when I don't use trizol, my lowry assay works well) - is there a quantitation assay which I can use if I have some phenol contamination?
I am sick of trial and error loading!
this might not help - but do you have to use Trizol? in my experience, Trizol is more trouble than it is worth for both RNA and protein! I would suggest changing your isolation protocol.
Hard to answer this question without any more specific information regarding what you've tried.
Also, I think you should re-post this question in the molecular biology section, rather than the immunology section. you'll get more answers that way...
It almost sounds as if the problem isn't with the Sybr. Are you sure your PCR mix is working? Did I miss this, or have you not gotten any products with this mix in the presence OR absence of Sybr? Could it be that the mix isn't working?
Perhaps you could try it with normal Taq and add a bit of Sybr to your master mix prior to adding to samples? I don't have any suggestions on concentrations - i've only ever used commercial kits.
Maybe try Nick's suggestion for some freebies... I've used Sigma mixes and Invitrogen mixes with success...
i just finished myself, and was FREAKING OUT for the majority of it. but i am glad i finished. and to my surprise i got offered 4 jobs without having to apply for anything! and no, i havent actually managed to publish anything yet so it's got nothing to do with publication record.
i'm thinking of trying out my current post-doc for a year or two, then heading off (i'm in australia). probably to europe. then coming back to sun, sand, and surf and doing a masters in pharmacy at university!
but stick with it julie - it'll be over before you know it. i think as a student you lose perspective and think that it has to be the most important thing in your life. but it's not!
it's much easier to quit a post-doc and do something else than it is to bring yourself to quit a PhD! a post-doc is JUST A JOB!
when you were designing the primers, did you follow the guidelines of the stratagene kit? it gives pretty detailed instructions. the Tm is supposed to be around 80 degrees anyway.
and did you get your primers PAGE of HPLC purified? it's supposed to increase the efficiency of the mutagenesis. dont know if this is true, but so far PAGE purification (Sigma-Genosys probes) has worked for me (although it is more expensive).
if background is a problem then there are several things you can do. first thing is look at the composition of your hybridisation and pre-hybridisation buffer. does it have blocking agents? such as sheared salmon/fish sperm DNA, and/or yeast tRNA, and/or Cot1 DNA?
Also, the annealing temp is important. often increasing the annelaing temp will decrease your background, but if you go too high then you may not get any annealing fo the probe. next thing to change is the washing temperature. the higher you go the less background (and signal if not careful) you will get.
does the DIG labelling protocol include a probe purification step? or do you add it directly to your northern after you label it? i would recommend an ethanol precipitation (with sodium acetate) to get rid of excess free nucleotides. although DO NOT do a phenol:chloroform step, as the DIG will separate into the non-aqueous phase, taking your probe with it! the cleaner your probe the better the hybridisation and signal.
you can also try pre-hybridising for longer to prepare your membrane and block any non-specific annealing.
and lastly, the cleaner the RNA prior to binding it to the membrane, the better the signal you will get. and i guess the amount of RNA you run on the gel will affect the signal. i work with animal tissue, but i normally run 10 to 15 microgrammes of total RNA for each sample.
Normally, miniprep kits come with an elution buffer that contains Tris and EDTA. These buffers can interfere with the sequencing reactions, and so I always elute in purified water prior to sequencing. I would recommed that you try this first, and if it doesnt work, then do the phenol chlorofom purification and precipitation.
Are you eluting in water or elution buffer?
i tried for AGES to get Amersham Cy5 to work, and people thought i was mad when i said it didnt. i called amersham on numerous occasions and they kept telling me that i was the only one that had reported the problem - although in all fairness they did replace the dyes twice. Cy3 has always worked for me though.
I ended up switching to AlexaFluor dyes (molecular probes - they sent me some free samples to try first). But i also found adding DTT to the washes helped a bit. i would also try and quantitate the labelling reaction (desribed in the amersham manual). i was doing in-direct labelling with amino-allyl-dUTP, so i dont know if direct labelling would work differently. also, i found that if you labelled more cDNA than the recommended amount ( i was using both amplified RNA and total RNA - around 10 micrograms of amplified RNA or 40 micrograms of total RNA. probably a bit high but it gave good results) the Cy5 seemed to work better.
so i cant make any sense of it. hopefully these suggestions help.
i figured eppendorf just wants you to buy more buffer, but i make my own anyway - someone just told me once that you cant reuse it! evidently, they were very wrong!
DNA is soluble in water because the water molecules intercalate into the phosphate backbone of the DNA and thus maintain it in a soluble state. When you add salt and alcohol you are essentially removing the water molecules and teh DNA then precipitates.
i just used google to do a search, and found the commercially available 50XTAE from eppendorf should only be used once. presumably its the same recipe as standard TAE...
"For agarose gel electrophoresis, 50x TAE should be diluted to a working concentration of 1x. The TAE buffer should be
replaced after each electrophoresis run due to the anode solution becoming alkaline and the cathode solution acidic, which
results in lowered DNA mobility."
but if it works, then may as well.
I'm not sure I get the question exactly, but have you cheched your pH for your buffers? Should be around pH 8.0? DNA tends to run smeared if the pH is too high or low, because pH can shear your DNA fragment.
Some loading buffers that have bromophenol blue and xylene cyanol act as pH indicators, alhtough I'm not sure at what pH they change. But if they are anything other than dark blue (for bromophenol blue) and light blue (cyanol), then you should begin to worry!
Hope this helps.
I've just started in a new lab, and here they re-use their 1X TAE for DNA gels up to 5 times. I was under the impression that it should only be used once, however TBE could be used around 3 times.
So, should you, or should you not re-use TAE?
there are several stains you can use on a northern membrane but i normally use a methylene blue stain:
Methylene Blue stain:
0.03% methylene blue
0.3M sodium acetate (pH 5.2)
Stain membrane for about 1-2 minutes (if stain is new – NO LONGER - don’t stain for too long if the stain is new or you’ll never get it out!). you should see bands appearing on the membrane – if not, you may need to destain it slightly because it may be too dark). You will see 28S and 18S rRNA bands (28S on top and 18S on bottom) if you work with animal RNA - not sure about other types of RNA. Smearing down the lanes will indicate RNA degradation. Bit of smearing is ok but too much is not. This will tell you how well your transfer worked. Dont let the stain dry out too much on your membrane before you destain or it may be hard to get out. You can leave the membrane soaking in water in the meantime (but not for hours and hours!).
You will need to destain before probing the membrane. The stain can be reused heaps of times (the older it gets the longer you may need to stain). You destain the membrane by successive washes in 0.1X SSPE
0.1% SDS (add SDS after the SSPE and water or it will come out of solution in the high salt content of the SSPE). Keep destaining until the membrane is clean again. Then rinse in water and dry the membrane or probe it straight away.
This should tell you whether the problem was the phenol in your RNA because if it was, then the transfer would not have worked very well. Another important thing to know is that you should never phenol extract a DIG-labelled probe after you make it. This is because the DIG will separate into the phenol phase and you wont be left with anything!
hope all this helps!