July

June 30th - July 6th

Testing the SPAAC reaction in general

In this step, the SPAAC reaction will be tested with just the unnatural amino acid pAzF and DBCO-PEG (~10 kDa). 

Phosphate buffer (100 mL, 20mM trisodium phosphate, pH 7.4):

-          100 mL demi water

-          1 tablet PBS (Phosphate Buffer Saline)

-          3,74 g KCl (potassium chloride)

-          500 µL Tween 20

Calibration curve and examination of the absorption wavelength of DBCO (306 nm)

First, the amount of required substance has to be determined.  This was done by filling the cuvette with consecutively 1.0, 1.8, 2.0 and 3.0 mL acetone. The settings of the measurement were as follows:

These measurements showed that 1.8 mL is sufficient to measure absorption.

Next, a stock-solution of DBCO-PEG (10 kDa) is made (142.9 µM):

-          4.5 mL phosphate buffer

-          6.43 mg DBCO-PEG (10 kDa)

The first measurement has been performed to determine the cut-off value of the buffer.  Hereafter, the concentration series of DBCO-PEG (10 kDa) are measured. The concentrations were subsequently 142.9, 100, 70, 49, 34.3, 24, 16.8 and 11.8 μM. The measurements were performed with the following settings.

With the obtained absorption spectra, a calibration curve can be made by fitting a line through the values for 306 nm. The slope of this line is the molar extinction coefficient and can be combined with the Beer-Lambert Law to determine the concentration of DBCO in the reaction mixture.

July 7th - July 13th

Deze week hebben we ook hele leuke dingen gedaan.