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I also store my working stocks of primers (10 micro molar) at -20C.
I've never had a issue with repeated freeze thaws for those primers I've used several times (I don't often use the same set of primers more than a few times, usually actually only once).
If I were you, I would make up my working stock of primers, and then store them in smaller aliquots so that the number of freeze thaws is limited. You could even store them in single use aliquots (say if you do 100 reactions a day, store just enough for this in each aliquot).
#143079 Primers Freeze-Thaw
Posted
on 10 October 2012 - 01:28 AM
#142667 DNA extraction from Fin Clips
Posted
on 02 October 2012 - 12:40 PM
The usual way of doing these sorts of things (usually used for genotyping) is to digest in proteinase K for a couple of hours then proceed with a crude DNA prep using salt and ethanol. I don't know how compatible this will be with the kit you want to use.
Grinding in nitrogen is pretty fast - you need to have some mortar and pestles or microuge tube grinders. Pour a little LN2 into the vessel, enough that the sample will freeze, then attack it with some vigour. Once ground, either scrape into an appropriate tube or add the lysis buffer and transfer to a tube. If you have any liquid (such as water or other buffering solution) around the tissue, it will form a solid lump which will be very hard to grind up, so make sure you dry off the fins before grinding.
Grinding in nitrogen is pretty fast - you need to have some mortar and pestles or microuge tube grinders. Pour a little LN2 into the vessel, enough that the sample will freeze, then attack it with some vigour. Once ground, either scrape into an appropriate tube or add the lysis buffer and transfer to a tube. If you have any liquid (such as water or other buffering solution) around the tissue, it will form a solid lump which will be very hard to grind up, so make sure you dry off the fins before grinding.
#142251 What else except for research?
Posted
on 25 September 2012 - 10:16 PM
So that's where you were 
Best of luck. Keeping my fingers crossed... and if they don't take you: Europe is always looking for good scientists 
Malaysia's loss is our win 
Best of luck. Keeping my fingers crossed... and if they don't take you: Europe is always looking for good scientists Malaysia's loss is our win
#141957 Help needed in reframing a sentence.
Posted
on 21 September 2012 - 04:13 AM
Sorry but I'm lost. You've got three different set ups? Because the last one sounds the same as the first one?
And do you mean one-way mirror?
"Pairs of fish were placed into tanks and separated by a one-way mirror for 5 minutes prior to recording interactions. Set ups were such that either only the big fish could see the small fish, or vice versa"
And I think it would be helpful to include a diagram (that's what I would do )
And do you mean one-way mirror?
"Pairs of fish were placed into tanks and separated by a one-way mirror for 5 minutes prior to recording interactions. Set ups were such that either only the big fish could see the small fish, or vice versa"
And I think it would be helpful to include a diagram (that's what I would do
)
#141894 How are Primers made?
Posted
on 20 September 2012 - 04:45 AM
you froward primer is your sequence as it is from 5' end to 3' end.
your Reverse primer is Reverse complement of your sequence.
Tm = 4(G + C) + 2(A + T) °C
Ions in solutions affect Tm.
For % GC you have to calculate number of G and C bases, combine them and divide by total number of bases.
O.D. is how much your absorbance of sample is. More the sample DNA more the O.D.
In other factors consideration is given to self priming, primer dimer sequence complementarity as non-desirable.
your Reverse primer is Reverse complement of your sequence.
Tm = 4(G + C) + 2(A + T) °C
Ions in solutions affect Tm.
For % GC you have to calculate number of G and C bases, combine them and divide by total number of bases.
O.D. is how much your absorbance of sample is. More the sample DNA more the O.D.
In other factors consideration is given to self priming, primer dimer sequence complementarity as non-desirable.
#141671 Buffer in Gel
Posted
on 18 September 2012 - 05:53 AM
I may run two gels if I am doing both, after loading fresh buffer. But I do not trust the buffer left over in a gel box others have used. I even wash the gel box out after I'm done (probably the only time it ever gets washed).
#141347 Difference between Cover letter & motivation letter
Posted
on 13 September 2012 - 12:48 AM
A motivation letter is the same as statement of purpose (SOP = for US universities).
So what should i write in the body of the email?
So what should i write in the body of the email?
#141320 Non-hazardous substitute for ethidium bromide?
Posted
on 12 September 2012 - 06:53 PM
Here are 2 gel photos. One gel has EtBr in it and the photo was taken on UV light box set at 302 nm. The other gel does not have any dye in it. Instead the DNA was loaded using 6x loading buffer containing 1/100 EZ-Vision stain. This gel photo was taken on UV light box set to 365 nm.
The 6x loading buffer consists of 1x TAE (pH 8.3), 50% sucrose w/v, 0.1% xylene cyanole and 0.1% bromophenol blue.
Add 10 uL of EZ-Vision concentrate (Amresco cat# N391-5MLDRP) to 1 mL of the 6x loading buffer. Keep in dark (use amber tubes). Remember to also use this for your size markers.
The 6x loading buffer consists of 1x TAE (pH 8.3), 50% sucrose w/v, 0.1% xylene cyanole and 0.1% bromophenol blue.
Add 10 uL of EZ-Vision concentrate (Amresco cat# N391-5MLDRP) to 1 mL of the 6x loading buffer. Keep in dark (use amber tubes). Remember to also use this for your size markers.
Attached Thumbnails
#141307 0.1 Volume
Posted
on 12 September 2012 - 01:42 PM
0.1 volume is 1/10th of the volume that you already have. e.g. if you are making up your ligation in 10 ul, 0.1 volumes would be 1 ul.
#141251 Non-hazardous substitute for ethidium bromide?
Posted
on 12 September 2012 - 06:27 AM
the dye in the loading buffer is a tracking dye. you use it to judge when to end the run. if you are losing the band during the run then it is probably diffusing and not due to the visualization dye. you may be able to see it if you look at the gel on a white background (we put white paper under).
#141253 1:10000X dilution
Posted
on 12 September 2012 - 06:33 AM
This is how I work it out:
Dilution factor is 40 000, volume of gel is, for example, 50ml
50ml/ 40 000= 0.00125ml (then x 1000 to give the volume in ul, so 1.25ul)
Dilution factor is 40 000, volume of gel is, for example, 50ml
50ml/ 40 000= 0.00125ml (then x 1000 to give the volume in ul, so 1.25ul)
#141163 What else except for research?
Posted
on 11 September 2012 - 06:28 AM
you can try for another post-doc position elsewhere or join an industry as a post doc
#141164 Non-hazardous substitute for ethidium bromide?
Posted
on 11 September 2012 - 06:30 AM
Is anyone using Gel red for gel electrophoresis?
I needed to ask something about this product so if anyone has any experience in using this then please let me know.
Moved and merged so as to keep recent discussion in one place. Bob
I needed to ask something about this product so if anyone has any experience in using this then please let me know.
Moved and merged so as to keep recent discussion in one place. Bob
#141152 TAE Vs TBE buffer
Posted
on 11 September 2012 - 02:37 AM
As leelee said TBE causes less heating because borate ions have better mobility than acetate. The problem with using borate is if you want to excise your band out of the gel and purify it the borate ions form complexes with the DNA and the yield is much lower. Therefore people use TAE. There was a paper on this subject a while ago called "Not your father's buffer" and it said that the best is Li-borate or Na-borate and in my experience you can really speed things up with a different buffer.
