Template:Team:Groningen/CONTENT/LOGBOOK/PCR pg31
PCR (pg31)
PCR was done to check if Gibson assembly was successful.
Make a more negatively charged biofilm.
Gel did not show the correct insert. Full PGA sequence ordered at IDT. Unfortunately delivery was due at September 17th. Too late for us to do experiments.
<a class="postscriptum protocol" href="https://2015.igem.org/Team:Groningen/Protocols_and_Protocols/PCR">PCR</a>
10:00, 10 August 2015 - 17:00, 17 September 2015
A PCR was done to determine the size of the Gibson product with the following reaction mixture and thermocycle.
Compound
Amount
Q5 polymerase
9 µL
Template DNA (Gibson product)
1 µL
Each primer
0.3 µL
\( \mathrm{H_2O}\)
9 µL
PCR components. Primers: prefix: CAGGCAGTTGAATTCGCGGCCGCTTCTAGA, suffix: CTTGAGCTCCTGCAGCGGCCGCTACTAGTA.
The following thermocycle was used for the PCR.
#
Step
Temperature
Time
1
Initial denaturation
98 °C
5:00
2
Denaturation
98 °C
0:30
3
Annealing
60 °C
0:30
4
Extension
72 °C
3:00
5
Go back to step 2 (30x)
6
Final extension
72 °C
10:00
PCR Thermocycle.
The PCR prduct was loaded on a 1% agarose gel with DNA stain G (1:50000) and run for 30 min on 100 V.
Sample:
1 µL DNA
1 µL 6x buffer.
4 µL \( \mathrm{H_2O}\).
Ladder:
2 µL Thermo Scientific GeneRuler 1kb DNA ladder, ready to use.
No correct insert was shown on the gel. Then the PGA DNA was ordered from IDT in one block in an ampicillin backbone. Unfortunately they had problems synthesizing it and delivery date was at the 17th of September. Too late for us to do experiments.
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