Butter coffee

Most people have heard of Bulletproof coffee.  It’s a sherpa-inspired tea drink that exploded in Silicon Valley a number of years ago.  The Bulletproof brand is “mycotoxin-free” coffee (???), medium chain triglycerides and butter. 

It appears as a green-yellow oil slick on my coffee

Butter is the bacon of dairy!  I don’t know if butter can make anything worse.  With my biases in mind I took the plunge.  I skipped the mycotoxin-free coffee and used my standard issue coffee.  It sounded like one-part hippie magic and one-part Wakefieldian science.  The roasting and brewing process destroys most, if not all mycotoxins.  I sliced off the equivalent of two tablespoons of butter into my coffee, mixed and then added water to the desired volume. 

What’s it like?  A delicious oil-slick.  The butter mostly forms a phase over the coffee.  The first few sips are decadent and rich – the butter tends to give my coffee a nutty flavor, not a negative quality. 

I don’t use this beverage in place of a breakfast; it is used as an addition.  I can’t  tell if this concoction makes me more focused.  It does seem to make me feel warmer and sweatier than a regular cup of joe.  The extra calories are saiting and my A.M blood sugar is even until 13:00.

That "fat white guy." – My journey from sugar – 2017-Jan-27

I am down 15 lbs since December 22nd 2016. 

So my blood sugar is very stable.  There is a period every afternoon, between 14:30 and 15:30 where my blood sugar dips and I feel sluggish and foggy.  I keep dry almonds and walnuts in my briefcase to ride out the sugar low. 

I am waiting until January 30th to have my blood work performed.  I am curious whether 6 weeks with this modified diet has any harmful side-effects on my vitals. 

The oddest observation that my daily step count has decreased despite the mass reduction.  As measured by my Iphone’s Health Data app.  My step count peaked on December 27th averaging 11,000 steps.  Since December 27th I am averaging 4,900 steps. 

Amplifying Large AT rich amplicons with Pfu type polymerases

This is a protocol for amplifying large amplicons that are high in AT. I developed it in order to amplify the 8.2 Kb region containing the promoter of Arabidopsis FT which is approximately 70% AT. In my hands it worked reliably with an 8 Kb amplicon (DeBono notebook 1, July 2010) but can be easily modified for longer products using more dNTP and optimized template concentration.

Methods

1. Very clean, homogeneous DNA. This protocol may work with genomic DNA but it is best to use DNA that contains many copies of your desired amplicon such as a BAC or a plasmid. I was using ~ 120 ng of BAC DNA.

2. A Pfu-like polymerase marketed as iProof, Phusion, Q5, and KAPA HiFi.

3. Low temperature 2-step PCR. 2-step PCR excludes the annealing step. The primers anneal to the template as the temperature drops to 64.5oC. Extension also occurs at 64.5oC.

Here is my 2-step PCR cycle:

98oC 60 s

98oC 20 s

64.5oC 90 s/1 Kb

Repeat steps 2-3 30-40 times

Polishing step at 64.5 for the amount of time specified in step 3.

This PCR regimen worked well for the Arabidopsis FT promoter as well as amplifying recalcitrant pectin methylesterases.

CPEC: Make multi-piece constructs with just cloning polymerase.

I love Gibson assembly.  It works almost everytime

(can’t help but put this here).

Gibson Assembly is much more reliable.

There are times when some assemblies do not work because of an issue with complementarity within the 3′ overhang that reduces chances for binding with the 3′ overhang in the matching DNA.  
When this situation arises I use a technique called circular polymerase extension cloning (CPEC)) by Quan and Tian.  The minimalism and likelihood for successful cloning is astounding.  In my hands I can make six more pieces work.

CPEC works by creating amplicons with overlapping 5′ and 3′ ends that are complementary to the 3′ and 5′ ends of neighboring DNA fragment(s).  The complementary ends have to be long enough to generate an annealing temperature greater than or equal to 60oC.  For a CPEC PCR, you assemble as PCR with your high-fidelity polymerase as usual.  Instead of adding primers and template to amplify, you add the fragments with end-complementarity.  In my hands 55oC annealing works nicely.  Then I perform extensions that are sufficiently long to circle the entire circularized vector; this works out to about 15 seconds / kilobase for Q5 polymerase.  At least 12 cycles are required.  That’s it.  Transform the product or examine it on a gel.  You’ll see a smear below and above a band corresponding to your fully circularized vector.  I was worried that using a polymerase would cause amplification errors. But there are no primers with which to create amplification errors.  Again, in my hands I have not observed errors as a result of CPEC.

That "fat white guy." – My journey from sugar – 2017 – January, 11.

A good friend recently let me know that her ex-husband referred to me as ‘that fat white guy.’  Darn.  I had an idea.  My activity level plummeted with the arrival of the twins and, if I’m completely honest, for 5 years prior to their births.    While on vacation at the end of last year I read an Aeon article entitled The case against sugar – A potent toxin that alters hormones and metabolism, sugar sets the stage for epidemic levels of obesity and diabetes .  The piece is a teaser for a book by Gary Taubes’ The case against sugar.  I’ve yet to read the book but it is on my radar.

The article is worth reading and pointed out an interesting concept that seems contrary to thermodynamics: ‘a calorie is a calorie;’ is incorrect.  I happen to have a project at work looking at the metabolism of single celled organisms. Modifying diet, modifying the sugar solutions we feed the microorganisms makes for completely different transcription profiles as measured by RNA sequencing.   If this happens in single celled organisms, it most certainly happens in humans.  Taubes makes the argument that sugar is toxic (in the quantities found in typical Western diets) because in many unfortunate souls, creates fat, through insulin resistance leading to diabetes – my ancestry has at least three diabetics.  Some however, are metabolically lucky; my partner can eat anything without worry of becoming fat.  When pregnant she had difficulty gaining weight and went through extraordinary measures to gain weight (daily lunchtime milkshakes and large daily protein portions). When the twins were born the weight disappeared without exercise.  There may exist some truth behind the thinking that a calorie is not a calorie and human metabolism is more complicated that the amount of energy that can cause water to rise 1 degree Celsius.

So why write about my expanding waist line?  A reason is to publicly declare I want to improve from being ‘that fat white guy.’  Lerner and Tetlock‘s review illustrates when goals are made public, people tend to push harder toward their achievement.

What have I done so far?  My weight has not plummeted precipitously now that I am avoiding sugar and white flour.  My blood sugar level is stable and I no longer have sugar-lows where I am unable to concentrate.  My last weigh-in showed a decrease of approximately 5 kilograms.  I am however fastening my belt with its tightest loop hole.  Tomorrow I go for a physical and will report back with information from my blood work and other tests.