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There have been 30 items by ros (Search limited from 20-April 20)



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#13318 Stable Transfections

Posted by ros on 31 March 2005 - 08:19 PM in Molecular Biology

hi all

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?

thanks



#13313 protein quantitation

Posted by ros on 31 March 2005 - 06:01 PM in Protein and Proteomics

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!  :(

<{POST_SNAPBACK}>



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.



#10860 Storage of Midipreps

Posted by ros on 07 February 2005 - 02:16 PM in Molecular Biology

Hi,

You should really store your plasmids in the fridge (4 degrees Celcius) because constant freeze-thawing can nick the DNA. However if you want long term storage, the -20 degrees is probably OK.



#10686 Biology Forum II

Posted by ros on 02 February 2005 - 07:57 PM in Chit Chat

nope! didn't like it. seems to be more about people asking general questions.

thanks for the suggestion though!



#10685 Is there anybody use Taq polymerase of Fermemtas

Posted by ros on 02 February 2005 - 07:40 PM in PCR Reagents and Equipments

works fine for most applications. havent tried it for anything bigger than 1.5 kb.



#10684 RT-PCR with B-cells as template

Posted by ros on 02 February 2005 - 07:38 PM in Molecular Biology

Hi Ali,

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...



#10683 Realtime RT-PCR problem - no product

Posted by ros on 02 February 2005 - 07:31 PM in Molecular Biology

I don't really get how you could have a melting curve without any amplification. doesn't the melting curve record a decrease in fluorescence as you increase the temp and thus denature the DNA (so Sybr doesn't fluoresce anymore...)?

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...



#10682 Aminoallyl Labeling

Posted by ros on 02 February 2005 - 07:22 PM in Molecular Biology

in my experience amino-allyl labelling works well, although Cy3 ALWAYS works better than Cy5.

there is definately a dye bias for Cy3 in preference to Cy5. but as long as you do reverse labelling controls, you should be fine.



#10681 I'm lost... What to do after PhD???

Posted by ros on 02 February 2005 - 07:06 PM in Career Advice

cool! pleased to "meet" you!



#10669 I'm lost... What to do after PhD???

Posted by ros on 02 February 2005 - 02:41 PM in Career Advice

newcastle, australia



#10625 I'm lost... What to do after PhD???

Posted by ros on 01 February 2005 - 06:28 PM in Career Advice

i think that if you can get a PhD in science, then you can do practically anything BECAUSE IT'S SO DARN HARD!

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!



#10623 Helping Post-doctoral fellows in life

Posted by ros on 01 February 2005 - 06:15 PM in Career Advice

another question - what continent do you have to be on?



#10618 Where are you from?

Posted by ros on 01 February 2005 - 02:28 PM in Chit Chat

only one from australia?!?!?!? ;)



#10617 northern blot probe preparation

Posted by ros on 01 February 2005 - 02:22 PM in Molecular Biology

even if you probe RNA with cDNA it's still called a northern - not a southern. I incorporate alpha-32P-dCTP using the Prime-a-Gene® labelling system from Promega. works really well and with high labelling efficiency...



#10616 site directed mutagenesis

Posted by ros on 01 February 2005 - 02:17 PM in Molecular Biology

i dont think your primer dimers should be affecting your colony number because they shouldnt contain the antibody selection gene that the whole plasmid contains, and hence should not be able to grow on antibiotic plates.

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).



#10615 DIG Northern

Posted by ros on 01 February 2005 - 02:12 PM in Molecular Biology

i've never done a DIG northern but i have lots of experience with radioactive ones (32-p labelled probes).

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.



#10614 Plasmid DNA cleaning

Posted by ros on 01 February 2005 - 01:59 PM in Molecular Biology

I think the most efficient way of cleaning your DNA would be a phenol:chloroform:isoamyl alcohol purification and then a sodium acetate/ethanol precipitation.

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?



#10565 Microarray dye balance

Posted by ros on 31 January 2005 - 02:34 PM in Molecular Biology

wow and i thought it was just me!

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.



#10564 Reuse TAE buffer and agarose gel?

Posted by ros on 31 January 2005 - 02:22 PM in Molecular Biology

so how do you reuse a gel? do you just run your samples off the bottom and then reload new ones? i take it you dont add ethidium bromide to the actual gel itself before it sets? how do you get rid of the ethidium anyway - whether you add it to the gel or whether you stain your gel afterwards?

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!



#10518 ucleic acid precipitation

Posted by ros on 30 January 2005 - 04:47 PM in Molecular Biology

ok i remember something about...

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.



#10441 Reuse TAE buffer and agarose gel?

Posted by ros on 27 January 2005 - 10:16 PM in Molecular Biology

hmm...interesting. thanks. 10 times - wow!!

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...

Website:
www.eppendorf.com/script/cms-newspic. php?id=1771&inline=1&col=DOWNLOADFILE

quote:
"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.

thanks again...



#10438 PAGE

Posted by ros on 27 January 2005 - 09:59 PM in Molecular Biology

HI

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.



#10436 Reuse TAE buffer and agarose gel?

Posted by ros on 27 January 2005 - 09:44 PM in Molecular Biology

Hi all,

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?

Thanks alot....

Ros[B][COLOR=red]



#6312 Does phenol affect Norhern blot?

Posted by ros on 02 August 2004 - 05:23 PM in Molecular Biology

i dont think that the DIG-labelled probe will degrade the RNA on your membrane. Once it is immobilised and bout to the membrane it should be ok for a while. When i do northerns, i don't always use RNAse-free reagents (eg. hybridisation buffers) with my membranes and they work well anyway. so i wouldnt worry too much about the DIG probe if it is synthesised by PCR.



#6231 Does phenol affect Norhern blot?

Posted by ros on 26 July 2004 - 05:25 PM in Molecular Biology

Did you do any kind of stain on the northern membrane defore you probed it? If you had too much phenol in you RNA then it could be a problem with your transfer onto the membrane. However, you should be able to get rid fo the phenol by ethanol precipitating the RNA prior to running it on the gel to probe.

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!
:unsure:




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