PNB 2265 lab 7 standard curve

what is a standard curve

A graph that relates:
» Known concentration → measured absorbance
» Used to determine unknown concentrations
» Example equation:
» Y = mX + b
» Where:
Y = absorbance
X = glucose concentration

x=

glucose concentration

y=

absorbance

why do we do this lab?

In Lab 8, you will measure glucose transport
across intestinal tissue.
But:
• Glucose cannot be measured directly
Instead we measure absorbance
• Therefore:
• We need a standard curve to convert
absorbance → glucose concentration

DNS is used

used as a proxy for glucose levels in our assay
• Reacts with reducing sugars and forms a product that absorbs light at 540 nm

DNS is

yellow colored

ANS

red brown

prepare standard solution

1st set (0,5,10,20,30, 50 mM of glucose)

2nd set (standard +DNS + H20)

boil

cool + 5 mL of H20

measure absorbance at 540 nm

if your R2 value is below

0.98 you must repeat the curve

how to use micropipette

When loading liquid
• Press the button to the first stop and then release the button
smoothly to the upper stop.
• DO NOT press all the way down to the second stop!!!
To release the liquid
• Press all the way down to the second stop.

cleanup for the lab

DNS is toxic – wear gloves, dispose in separate waste
containers.
• Pour DNS waste liquid inside the funnel (not the lid).
• Rinse the tubes with tap water and put them upside and
down.
• If waste is full – notify TA immediately.
• Ensure your hot plate is OFF.
• Wipe down and organize your bench to how you found it

what can you do to improve the fit of the curve

clean all the test tubes with a Kim wipe if still does not work remeasure the one that is off

trachea vs esophagus

esophagus > stratified squamous epithelial cells

trachea > ciliated pseudostratified epithelium; hyaline cartilage rings

accessory organs

liver

gallbladder

pancreas

salivary glands

order structure of the GI tract from smallest to largest

smallest > microvilli

villi

plicae circulares

largest is rugae

all solutions prepared should add up to

1000 ul

Why do you need an R² value above 0.98? What should you do if you have an R² value below 0.98?

You need an R^2 value above 0.98 because you use it to determine the glucose concentration in the experimental samples which is the intestinal sacs, which also helps with predictive validity. If your value is below 0.98, wipe down the samples with a Kim-wipe, check for outliers, improve technique, and redo samples that are far from the line of best fit, if one is off, redo at least two samples that you made to determine the curve. 

Use your standard curve to calculate the glucose concentration of a sample with 0.11 absorbance reading. Show your math here for full credit

Glucose concentration would be 53.6 mM. 

Equation y=(1.97×10−3)x +(4.40×10−3) solving for x, as y=0.11

0.11= (1.97×10−3)x +4.40×10−3

0.156/ (1.97 x 10^-3)

x= 53.6 mM

mucosa

the innermost layer that consists of the following:

consists of

epithelial lining

lamina propria

muscularis mucosae