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Axolotl9250

Member Since 04 Jul 2012
Offline Last Active Apr 03 2013 07:50 AM

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22, PhD Student at UEA and Sainsbury Lab studying Plant Pathogen Evolution. Avid science geek, gamer and proud of it. Opinions are my own.

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Evolutionary Genomics and Biology of Plant Pathogen Effectors.

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tookta103
08 Jul 2012 - 09:22

Posts I've Made

In Topic: Making a buffer

05 September 2012 - 02:18 AM

I see, if I need the pKa of tartaric acid, I've got the problem that 4 different values are given:

L(+) 25 °C :

pKa1= 2.95

pKa2= 4.25

meso 25 °C:

pKa1= 3.22

pKa2= 4.85

The natural form is L-(+)-tartaric acid or dextrotartaric acid. So is that the pKa to use? With the phosphate buffer I used a pKa close to the desired pH for the calculation, so if this applies then I should use pKa2 of 4.25 rather than pKa1 2.95?

In Topic: Making a buffer

04 September 2012 - 07:06 AM

I see a answer on a web page - someone asked a similar question and got the answer to simply dissolve the sodium tartrate into water at the molar concentration desired and adjust the pH with HCl or NaOH and pH meter. So now my question is why is it for some buffers - the preparation is the precise combination of two things, like the sodium phosphate buffer, and for some buffers people simply weight it out, dissolve it and adjust the pH? Can you in reality do either method for any buffer or is it only suitable for some?

In Topic: CTAB of Fungi

30 August 2012 - 02:47 PM

This is awfully strange, do primer dimers often mean no desired amplification? I would have thought I'd get them with some amplification. I did the same thing the previous PCR attempt and got desired amplicons and a bit of smearing so this time I was using different template amounts of 20ng 50ng and 100ng to compare them. The only thing I think I did differently was I added my primers to the buffer and dNTPs before template (on ice). The last time I dotted my primers and taq onto the side of the tube (I still did it with taq) so as upon heating they fall into the buffer, template & dNTPs and start the reaction. People have described making master mixes with primers in though so I didn't think it mattered much.

In Topic: CTAB of Fungi

30 August 2012 - 07:38 AM

I did my PCR and ended up with a single bright band (even in NTC), very small - near the level of the last band of my 100bp ladder. Am I looking at contamination or primer dimers? Absolutely nothing else was amplified and there are no smears. Just this one band in every lane. If so then I think I'll have to convince the two I'm working with the primers are no good and we need to try something else. I did everything in a flow hood I cleaned with a DNexitus plus solution, pipette tips straight from the autoclave too, sadly my pipettes are not autoclavable.

In Topic: CTAB of Fungi

27 August 2012 - 12:04 PM

I've tried another PCR reaction and it has worked this time, albeit I need to re-run it because I got some smear in my negative control. However the expected RAMS bands predicted to appear based on a previous student's experience did appear. I'm not entirely sure why the first reaction did not work, perhaps DNase, but if it were in my reagents then I would have expected the run that worked to fail as well. I have noticed I have a tendency to over-dry my DNA, but after a day or so of heating for a few minutes and then mixing sorts this out. My next goal is to try and vary the component amounts to reduce some of the noise and get an optimal result.

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