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Latest revision as of 21:41, 21 November 2015
PCR ferritin construct (fr1)
Amplifying ferritin
Development of ferritin
Ferritin was amplified successfully
<a class="postscriptum protocol" href="https://2015.igem.org/Team:Groningen/Protocols_and_Protocols/PCR">PCR</a>
00:00, 31 Augustus 2015 - 00:00, 31 Augustus 2015
Ferritin was ordered with sequence with prefix and suffix primers from IDT. PCR was performed (Figure 1) using NEB Q5 High-Fidelity 2X Master Mix with the prefix and suffix primers.
Primer
Sequence
prefix
CAGGCAGTTGAATTCGCGGCCGCTTCTAGA
suffix
CTTGAGCTCCTGCAGCGGCCGCTACTAGTA
Primers for ferritin PCR.
The following PCR mix was prepared.
Compound
Amount
Q5 polymerase
9 µL
Template DNA (Ferritin IDT)
1 µL
Each primer
0.3 µL
\( \mathrm{H_2O}\)
9 µL
PCR components.
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
1:30
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 1 hour on 100 V.
Sample:
1 µL DNA (ferritin).
1 µL 6x buffer.
4 µL \( \mathrm{H_2O}\).
Ladder:
2 µL Thermo Scientific GeneRuler 1kb DNA ladder, ready to use.
A band of approximately 1600 bp was visible on the gel. This corresponds to the size of ferritin.