Team:TU Eindhoven/Notebook
From 2014.igem.org
Line 3: | Line 3: | ||
<html> | <html> | ||
<head> | <head> | ||
- | |||
- | + | ||
- | + | ||
- | + | ||
- | + | ||
- | + | ||
- | + | ||
<link rel="stylesheet" type="text/css" href="https://2014.igem.org/Team:TU_Eindhoven/Notebook-css?action=raw&ctype=text/css"/> | <link rel="stylesheet" type="text/css" href="https://2014.igem.org/Team:TU_Eindhoven/Notebook-css?action=raw&ctype=text/css"/> | ||
<link rel="stylesheet" type="text/css" href="http://netdna.bootstrapcdn.com/font-awesome/3.2.1/css/font-awesome.css?action=raw&ctype=text/css"/> | <link rel="stylesheet" type="text/css" href="http://netdna.bootstrapcdn.com/font-awesome/3.2.1/css/font-awesome.css?action=raw&ctype=text/css"/> | ||
- | |||
- | |||
- | |||
- | |||
- | |||
- | |||
- | |||
</head> | </head> | ||
Revision as of 12:19, 15 July 2014
Notebook
Hallo hallo
February
March
April
May
June
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:
Photometric mode |
Abs |
Response |
Medium |
UV/Vis bandwith |
1.0 nm |
Scan Speed |
40 nm/min |
Start |
340 nm |
End |
190 nm |
Data interval |
0.5 nm |
Vertical size |
Auto |
Scan mode |
Continuos |
Accamulation/cycle |
Accamulation |
No. Of cycles |
2 |
Temperatuur |
20 ˚C |
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.
Photometric mode |
Abs |
Response |
Medium |
UV/Vis bandwith |
1.0 nm |
Scan Speed |
40 nm/min |
Start |
340 nm |
End |
220 nm |
Data interval |
0.5 nm |
Vertical size |
Auto |
Scan mode |
Continuos |
Accamulation/cycle |
Accamulation |
No. Of cycles |
2 |
Temperatuur |
37 ˚C |
Stirrer |
150 rpm |
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.
August
September
October