Team:Groningen/Notebook/tasA Gibson Assembly t7
Blue Bio Energy
tasA Gibson Assembly (t7)
A Gibson assembly was performed to assemble the PCR products of the tasA gene.
Our goal is to overexpress TasA by adding a second copy of the tasA gene under the control of a salt inducible promoter. This overexpression will lead to a stronger biofilm when the biofilm is exposed to salt water.
The Gibson assembly was performed.
Gibson assembly
00:00, 19 May 2015 - 00:00, 19 May 2015
For the Gibson assembly, DNA was used with the concentrations indicated in Table 1. The concentration in the second column is the concentration of DNA based on the gel. The concentrations listed in column 3 are obtained by using Nanodrop.
PCR product
Estimation (based on gel)
Nanodrop
1.3 (100 bp)
250 ng/µL
37 ng/µL
1.4 (150 bp)
170 ng/µL
27.1 ng/µL
2.5 (700 bp)
200 ng/µL
67.7 ng/µL
2.6 (250 bp)
40 ng/µL
42.1 ng/µL
DNA concentration of samples.
Two Gibson reaction samples were made.
#
Component
Amount
1
PCR product 1.3 (100 bp)
0.588 µL
PCR product 1.4 (150 bp)
0.260 µL
PCR product 2.5 (700 bp)
0.143 µL
\( \mathrm{H_2O}\)
4.00 µL
2
PCR product 2.5 (700 bp)
0.588 µL
PCR product 2.6 (250 bp)
0.893 µL
\( \mathrm{H_2O}\)
3.59 µL
Gibson reaction samples.
15 µL of Gibson reaction mixture (Clement) was added to a total volume of 20 µL and incubated for 1h at 50 °C.