Template:Team:Groningen/CONTENT/LOGBOOK/Colony PCR fr7
Colony PCR ferritin (fr7)
Colony PCR from ferritin transformation
Development ferritin
Positive colony
<a class="postscriptum protocol" href="https://2015.igem.org/Team:Groningen/Protocols_and_Protocols/ColonyPCR">Colony PCR</a>
17:00, 1 September 2015 - 21:00, 1 September 2015
Transformation was checked. On the plate were 5 white colonies visible. All these colonies were checked for the correct insert with a colony PCR. Each colony was taken from the plate and resuspended in 20 µL sterile MQ water. A Mastermix for 16 samples was made.
Component
Amount
Dreamtaq polymerase
3 µL
10x Dreamtaq buffer
30 µL
DNTP MM (2 mM)
30 µL
\( \mathrm{H_2O}\)
237 µL
Colony PCR primers
5 µL each
Components Mastermix. Colony PCR primers: forward: TCACGAGGCAGAATTTCAGATAAAAAAAAT reverse: ATCTTTTCGGTTTTAAAGAAAAAGGGCAGG
For the PCR samples, 18 µL of Mastermix was added to 4 µL of colony suspension. The following PCR thermocycle was used.
#
Step
Temperature
Time
1
Initial denaturation
98 °C
10:00
2
Denaturation
98 °C
0:30
2
Denaturation
98 °C
0:30
3
Annealing
55 °C
0:30
4
Extension
72 °C
2:00
5
Go back to step 2 (repeat 30x)
6
Final extension
72 °C
10:00
PCR thermocycle.
The samples were run on a 1% agarose gel with DNA stain G 1:50000. 5 µL of sample DNA was mixed with 1 µL 6x buffer. As a ladder 2 µL of the GeneRuler™ 1 kb DNA Ladder was used. The gel was run for ∼30 minutes at 100 V. All five colonies have the correct insert size.