Mixed Level Yoga at Burlington Yoga

By far my favorite yoga location – Burlington Yoga – I managed to get to the 7:30-9:00p, Mixed Level class on Friday.

I should have been posting this earlier but life was full of Stuff so I couldn’t. Mostly productive.

This was my first 1.5 hr class I ever attempted. Although I was satisfied by the moves enforced on me I did get impatient as an hour approached and I knew class was only half over. While everyone else appears to relax I seem to be the only one who seems to be incapable of absorbing calm most of the time and only be able to absorb greater intensity. I would intermittently pay attention to my breath when reminded but still could not focus on it otherwise. If I ever happen to find myself on adderal again, I would be curious to see if I could focus enough to pay attention in a yoga class.

One interesting aspect of the yoga that day was the frequent adjustments and massages the instructor provided. Apparently that is normal in Restorative yoga classes but I found it confusing since this wasn’t announced at the beginning of the class at all. Undefined variables give me more to think about and expound on in my mind which isn’t good.

There is a Mixed Levels class from 10:00-11:30am on Sunday next week. Since we will have gotten back from Plattsburg the day prior and be destroyed I think it might be a good idea to attempt that. I know that I need to do my yoga for this week though and I intend on aiming at Anusara Yoga at Patrick Gym on Wednesday.  I really am going to have to be sure to be on top of things.

Chromatography – Science Blog for 09-01/09-02

Science Blog for 09-01/09-02

I was considering continuing on with what my students in Adv Genetics will be doing but, to me, it’s a bit too simple. The phenol/chloroform was relatively simple but their next task will be to run a gel and I think I can skip that one.

Instead I thought I would touch on some of the topics my Biochemistry course is inflicting upon us. I thought Chromatography would be perfect. There are a LOT of type of Chromatography so I can make a relatively simple summation of the lot of them while essentially counting it as Biochem study as well.

 

Chromatography can be described as a type of laboratory techniques (of various flavors) in which a solution is fractionated or separated out based on some property.

  • Mobile Phase: The mixture of substances dissolved in a liquid that will be fractionated
  • Stationary Phase: a porous solid matrix in which the liquid will be passed through, often coupled with binding components

 

More vocabulary:

Partition Coefficient: the ratio of concentrations of a compound in a mixture; measure of the difference in solubility of a compound in two immiscible phases at equilibrium (represents stationary and mobile phases)

Analyte: target substance to be separated

Chromatogram/Chromatograph: a graph in which the x-axis represents retention time and the y-axis is a signal (from a spectrophotometer or mass spec) in which the signal is proportional to the concentration of the specific analyte separated

Eluate: the mobile phase exiting the column

Eulent: the solvent that carries the analyte

Retention Time: the time it takes for a particular analyte to pass through the system

 

In Column Chromatography (the only one I know anything about), the stationary phase exists in a tube (column) filling it entirely [packed] or concentrated on or along the inside of the tube well [open tubular column]. Solutes flow through the column as the mobile phase and are slowed due to interactions with the stationary phase. With a long column, portions of the mobile phase that have higher rates of migration will be separated first, followed by those with slower rates of migration.

Gel Filtration/Size Exclusion/Molecular Sieve Chromatography

This is a common form of chromatography in which the analyte is separated according to size and shape. In this case the analyte does not have interact with the stationary phase chemically/biochemically but only physically. This method is based on the ability of the analyte to penetrate and a pass through the gel bead stationary phase. Large molecules will elute from the column quicker than small particles that pass through pores of the gel beads. The smaller molecules will require a larger amount of eluent to elute.

Ion-Exchange Chromatography

In this form of chromatography charged molecules bind to oppositely charged groups that have been attached to the stationary phase matrix. Anions will bind to cationic groups (anion exchangers) and cations will bind to anionic grounds (cation exchangers). Polyelectrolytes, which are polyionic polymers with both positive and negative charges, will bind both cation or anion exchangers.

The binding affinity of the protein passing through can be complicated by the presence of other ions that may compete for the binding along with pH which will influence the protein’s net charge depending on the protein’s pI (Isoelectric point – point at which it has a net zero charge profile). Proteins that successfully bind can be eluted by an eluent with a higher salt concentration (salt = ions) or a pH that reduces the affinity with which it is binding the cation/anion exchanger.

Hydrophobic Interaction Chromatography

In this case the stationary phase matrix possesses octyl, butyl, or phenyl groups. These hydrophobic groups will interact with the hydrophobic groups of the proteins passing through. A buffer with high ionic strength, usually ammonium sulfate, is first passed through which reduces the solvation of the solutes and permit hydrophobic regions to be adsorbed. The high salt conditions promote hydrophobic interactions and aggregations. The greater the hydrophobicity of the molecule the less salt is required for promoting binding and the salt concentration can be gradually decreased to elute from the column in order of increasing hydrophobicity. Concentrations of detergent, which will disrupt the hydrophobic interactions, or changes in pH can also be used to elute.

Affinity Chromatography

My ‘favorite’ due to the fact that it is so specific, in this case a ligand attached to the matrix will specifically bind to a protein while all other proteins are washed out of the column. Elution conditions will have to be adjusted to release the binding. One thing to consider is that the binding has to be weak enough to be disabled (so biotin-streptavidin is probably not a good idea). Immunoaffinity uses Abs for this purpose. Metal chelate affinity involves diavalent metal cation (Zn2+) or (Ni2+) is attached so proteins with metal chelating groups (His tags) can be retained.

I am now distracted so I think that is good enough J

BYeee!

Yoga at UVM and I have been so good >:]

Alright alright!!

Went to Yoga at UVM. 7:45-8:45. Now, I will have to say right off the bat I have been manic-y for days. At first I thought it was the coffee mixed with whey. Now I am pretty sure the protein burst is partially to blame (and a nervous breakdown?). I have lost of a lot of weight but hadn’t adjusted my protein intake for a while. 10lbs since last year…kinda crazy…since I didn’t think I calculated that. Whatever whatever- yoga!!

So, as usual, I hated it at first. The room was INCREDIBLY PACKED since it is ‘free week’. Thank god it won’t be like that any other time. I could scarcely hear her yelling at me to be calm and think happy thoughts. The moves seemed rather erratic since nobody else could hear her either. And the pop music was a bit…out of alignment. We had to sit and close our eyes to the latest and greatest dance music.

It DID get serious with some balancing moves and a new move: “peaceful warrior” which is like warrior pose with the arm up. Maybe it’s not new (to me even) but now I remember the name for some reason.

There were definitely some strength-challenging moves. And I’m so sorry I am failing to describe yoga, really. But at least I am going, right?

Also, all this forced-meditation!! While manic too! Think happy thoughts, clear your mind. lol. Right, right, right…But guess what, I did do some thinking. Like I would if I went on a long bike ride. I haven’t taken adderal to calm down (yes, it would calm me down in the proper dose) and I HAVE NOT gone looking for affection/attention though I feel such dire need for it. So good, I am. Hah. So, I decided. I have been trying to calm myself for a long time. With CB being so erratic I took it upon myself to try to be as calm as humanly possible, even taking adderal so I could be calm around him (so that I didn’t do/say anything he might freak out about – even if I thought they were innocent comments/Qs). But that’s not me, really. And right now I feel like myself again. Sure, I can’t help it…unless I took drugs. But I’m not this time. I am done with all that. I was meant to be manic sometimes. I was meant to go insane sometimes. I’m totally okay helping out other people who are going insane, but if I don’t have my fair share and am not equally helped…beyond it not being fair, it suppresses my reality- and it forces me into that degraded state where I am trying so hard to continuously suppress myself. I need copious amts of affection and I need to give it too. Suppressing myself gets me less and allows me to give less. So if I wanna drop cauliflower on a random person’s desk I’m gonna do it. And when I finally get some love, I’m gonna love wicked hard and I better get all that back and if I need to cry I am not going to roll over and try to stifle it like I have been.

Isn’t that a better yoga post? Practically no yoga. Mission complete though. And maybe it Does count as yoga material since it was a post on my forced meditation. I also thought about a lot of other things too but I don’t wanna waste too much space.

I will be attempting to go to more UVM yoga classes in the future as they are so close by. Maybe I will be able to hear the instructor and learn what I am doing.

I actually did pretty good comparatively but the door next to me with a nice solid handle MAY have helped…

DNA/RNA Purification via Phenol/Chloroform Extraction

So instead of continuing with the GAL4 system I am going to deviate with the purpose of describing methodologies that directly relate to either the class (Advanced Genetics) that I am TAing or Biochemistry (which requires my memorization of a large variety of techniques).

 

I will start on the easy side and describe Protein & DNA/RNA Purification via Phenol/Chloroform Extraction. This is one of the first labs that I will be teaching to the Adv. Genetics students so I must be certain that I completely understand it myself.

 

Vocabulary [some of this will not be in the immediate phenol/chloroform description but is affiliated with previous collection protocol in which crude cell lysate is collected]:

Potassium Acetate: (CH3CO2K) A potassium salt of acetic acid whose purpose is to precipitate dodecyl sulfate (DS) and DS-bound proteins, which permits the removal of proteins from DNA

Sodium Dodecyl Sulfate: an amphiphilic anionic surfactant; it is a detergent used in lysing cells and denaturing proteins by disruption of noncovalent bonds

TE Buffer: buffer solution containing Tris (pH buffer) and EDTA (chelates cations) whose purpose is to solubilized DNA while protecting it from degradation

  • pH is usually adjusted to 7.5 for RNA and 8.0 for DNA

Phenol/Chloroform extraction is purposed for the removal of proteins from DNA samples. During the process two phases are developed called the aqueous phase (water-soluble) and the organic phase (slightly heavier, less-water soluble phase where lipids congregate). Between the two phases is an interface where proteins typically accumulate (having amphiphilic properties –  that is, both hydrophobic and hydrophilic portions).

Notes on Phenol & Chloroform:

  • Phenol must be at pH 7.5; this is important as chromosomal DNA will end up in the phenol phase if the pH is acidic (*why)
  • Phenol can undergo oxidation and will appear a yellowish/redish color instead of clear in this case; this can be damaging to DNA
    • (but probably not incredibly damaging since the protocol for the students doesn’t appear to address it but Does mention the yellow color change as an advantage for recognizing the interface –  since the proteins accumulate in the phenol phase – I would guess that despite its danger to DNA it typically isn’t inundated with it since the DNA fancies the aqueous phase)
    • Both Phenol and Chloroform are solvents for plastics, hence the requirement for glass pipets
    • Purified Phenol has a density of 1.07 g/cm3 and forms a lower phase when mixed with H2O (1.00 g/cm3).
      • Chloroform assists with the phase separation in that it is miscible (can mix) with phenol and possesses the higher density of 1.47 g/cm3.

The reason for DNA being miscible in the aqueous phase is due to its polarity; DNA is polar due to the negatively charged phosphate backbone. In the case of proteins; the various charged/uncharged groups with hydrophobic cores exposed (in the case of denaturation) will precipitate at the interface of the aqueous & phenol phases.

Other Notes:

  • Ethanol Precipitation often following Phenol/Chloroform extraction for the purpose of concentrating the DNA in a preferred buffer

Not too bad, right?

Gentle Hatha Yoga

Finally – this week = success. I threatened myself with having to go to Midnight Kripalu again and IT WORKED! Yet again, I ended up at Vermont Yoga (if it wasn’t clear, that was where I was for midnight Kripalu as well) and once again I was the only one there. While clearly the general population thinks fairly poorly of Vermont Yoga (there never being anyone at the classes) it is somewhat of an advantage to me in that I get to know the instructor fairly well and they cater the class to my level and can directly educate me personally.

The instructor was far more competent this time. The instructors at Kripalu seemed to fail at basic math (not being able to subtract $12 from $20) kind-of threw me off. She was serious about yoga and the movements while being considerate of other perspectives. She would describe fellow yoga instructors as varying between serious and light-hearted with both being admirable.

Beyond psychological perspectives of the instructor however- the class was better than I expected. I was a little worried that with “Gentle” in the name it might be TOO gentle. It started off a bit slow with very simple movements as most yoga does. The simplistic movements seemed a little more elongated in this class (likely due to it’s nature as ‘gentle’) but it moved on to deeper stretches and some challenges of strength (in the form of lunges of course). Because I was so new, the instructor was happy to go over the basic sun salutation with me in multiple versions.

I also noticed a ‘true mirror’ in the classroom. Just a side nugget of coolness.

You probably can tell these posts aren’t very descriptive. Don’t worry. The more yoga experience I get the more I will remember names of moves and be able to describe them. I could probably describe a downward dog at this point but I’d like to be able to describe more than the number of ways your can place your butt in the air so I am holding off until I think I am capable.

True meaning of this post: one more week of yoga success. Next week I will likely be trying the completely undefined “Yoga” at UVM so I won’t even be able to go on descriptions like ‘Kripalu’, ‘Kundalini’ or ‘Hatha’. I have hope that they will describe what kind of yoga it will be once I arrive. (Assuming I can achieve my goal next week…but with the threat of Midnight Kripalu I think I just might)

Part I: GAL4/UAS System in Drosophila melanogaster

Because it will take me far too long when I have excessive amts of other work to do to accomplish some of my technique descriptions I have decided to split them into parts at times.

GAL4/UAS System in Drosophila melanogaster

Part I: Vocabulary, Background (via vocab), & ‘Basic Idea’

Part II: Modifications to the GAL4/UAS system & Example of Use

Part I: Vocabulary, Background, & ‘Basic Idea’ of GAL4/UAS System in Drosophila melanogaster

Vocabulary

Enhancer: a region of DNA that can be bound by proteins to enhance the transcription of a gene cluster located either upstream or downstream of the gene it regulates.

Enhancer Trap: a technique used to identify enhancers in which there is a mobile element (usually P element) for the purpose of random insertion and a reporter gene.

  • P[lacZ] and P[GAL4] are the most common and basic enhancer traps
  • If the reporter (lacZ, or GAL4) integrates near an enhancer, it’s expression will reflect the expression pattern driven by that enhancer so that, with the increased expression, the site around the site of integration can be cloned out and identified.

P-element: a transposon (sequence can change its relative position on its own within a genome, also known as a ‘jumping gene’) that encodes for P transposase which recognizes a 31bp terminal inverted repeats of the P-element facilitating its own excision/re-insertion.

  • The random insertion can interfere with genes (by disrupting their sequence) or carry an additional gene of interest
  • The coding sequence for transposase and the recognition sequence for transposase (P Plasmid), of the P-element, can be separated to allow control over transposition
    • P plasmids contain a reporter gene (usually red-eye marker) and transposase recognition sequences along with gene of interest

GAL4 gene: encodes Gal4, yeast (Saccharomyces cerevisiase) transcription activator protein of 881 amino acids that regulates GAL10 & GAL1 (genes responsible for galactose metabolism)

  • Target sequence is a 17-mer

GAL80: protein that can repress GAL4 activity except under conditions when galactose is the only carbon source.

  • Binds the transactivation domain of GAL4 and prevents it from activating transcription
  • When expressed ubiquitously under control of tubulin 1alpha promoter – GAL4 activity can be suppressed in all tissues

UAS (Upstream Activation Sequence): a sequence possessing 4 copies of the GAL4 target found upstream of GAL1 & GAL10

One transgenic line is considered the ‘driver’ and possesses GAL4 expression in a known temporal & spatial pattern.

  • Temporal Pattern: GAL4 activity can be driven by expression from a heat-shock promoter (however, offers ubiquitous (not tissue-specific) transgene expression).
    • Additionally, in flies, there is an innate temperature dependence in which there is very little activity ~16C while ~29C provides maximum activity.
  • Spatial Pattern: Cell or Tissue determined by GAL4 driver specificity

Another transgenic line is required considered the ‘responder’ and possesses a UAS-dependent transgene.

The separate transgenic lines produce progeny with both GAL4 and UAS in which GAL4 now is capable of binding the UAS region and activating the affiliated transgene.

So, essentially, the GAL4/UAS system is a way of GAL4 that can be inserted randomly (using a P-element plasmid) in which the GAL4 is capable of activating UAS-gene of interest when the two separent parents possess the promoter/driver-GAL4 in one and the UAS-gene of interest in another mate and produce offspring possessing both the GAL4 and UAS-gene of interest together. It can be influenced by tissue-specific promoters so that the GAL4 is restricted to expression in particular regions of the fly so that that the UAS-gene of interest may only be expressed in that region. Even if UAS-gene of interest is present in every cell of the fly it will only be expressed with the presence of GAL4, which may be restricted.

References:

Elliott, D. A. and A. H. Brand (2008). “The GAL4 system : a versatile system for the expression of genes.” Methods Mol Biol 420: 79-95.

Duffy, J. B. (2002). “GAL4 system in Drosophila: a fly geneticist’s Swiss army knife.” Genesis 34(1-2): 1-15.

And, yeah, Wikipedia…

Midnight Kripalu

The Kripalu Center

That’s right. I went right out an accomplished my task for this week. Good thing too since this week only had a couple days left from when I announced that goal.

I will admit, when going here (and really, up until now) –  I was thinking of Kundalini which is my ‘favorite’ yoga, perhaps due to my liking not being upside down and all the core/arm work works around my weakness (balance) to an extent.

Throughout the class I was surprised at how unlike my last Kundalini class it was. My last class being over a year ago at a Healthworks in Cambridge of course. Kripalu turns out to be a form of Hatha yoga, but has a separate set of moves.

One of my favorites was something like leaping like a frog in place. As you can imagine, this was quite different from the slightly less dynamic upper-body oriented Kundalini I had been expecting. My mistake- I just assumed there was only one yoga that started with “K”.

Anyway, the instructor was quite friendly and I was the only soul present so it is possible that she went easy on me. Breathing is a challenge for me, as is balance. The breath movements in yoga are simply uncomfortable and, I feel, unnecessary. During Kripalu they actually seemed a lot more out of place and variable than in other yogas. Breaths had to be increased to be in line with the changing dynamics and pace of the movements.

Since this class is at 11pm on Thursday I might be going to it again. With UVM starting up though I am much more prone to save the $12 and go to UVM’s generic yoga classes. Though I hope I may still be able to find a Kundalini class somewhere.

I have added a link to the Kripalu Center which apparently was, at one point, an essentially religious cult. As is typical, their extremely over-paid leader had sex with various followers and eventually was forced to step down due to corruption. And I bet you thought yoga was so pure. Well, no, if you’re reading this…probably not. Most serious yoga-goers are absolute snobs and I would feel they are best described as ‘pricks’. I don’t really know much about that insult but I feel like it fits. But those such people might benifit from a taste of reality. It exists in all your subjects, world.