Template:Team:Groningen/CONTENT/EXPERIMENTS/Checked Growth
12:00, 13 August 2015 - 15:00, 13 August 2015
Designed a new suffix primer for bslA (yuaB) with the correct sequence for BioBrick compatibility
12:00, 31 August 2015 - 00:00, 31 August 2015
PCR bslA sequence with yuaB forward 2 primer and new suffix primer (figure 1) using NEB Q5 High-Fidelity 2X Master Mix.
Sequence
yuaB forward 2
GCGAATTCGCGGCCGCTTCTAGAGTTAGGGGGAATTTTGTTATGAAACGC
iGEM yuaB reverse 2
CGCTGCAGCGGCCGCTACTAGTATTAGTTGCAACCGCAAGGCTGAG
Primers for bslA PCR.
The following PCR mix was prepared.
Compound
Amount
Q5 polymerase
9 µL
Template DNA (Y4 DNA)
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 (bslA).
1 µL 6x buffer.
4 µL \( \mathrm{H_2O}\).
Ladder:
2 µL Thermo Scientific GeneRuler 1kb DNA ladder, ready to use.
Band of approximately 600bp was visible on the gel. This corresponds to the size of bslA.
15:30, 31 August 2015 - 16:00, 10 August 2015
PCR purification was done of PCR product and eluted in 21µL elution buffer. The concentration of 69,8ng/µL was determined using the Nanodrop. Product was stored as: yuaB, 31-8.
16:00, 31 August 2015 - 18:30, 10 August 2015
Digest bslA and pSB1C3 in order to ligate them. Samples were digested for 2h at 37°C.
#
Component
Amount
1
bslA
10 µL
10x buffer (2.1)
2 µL
PstI
1 µL
EcoRI
1 µL
\( \mathrm{H_2O}\)
6 µL
2
pSB1C3
10 µL
10x buffer (2.1)
2 µL
PstI
1 µL
EcoRI
1 µL
\( \mathrm{H_2O}\)
6 µL
Components for digestion.
18:30, 31 August 2015 - 20:00, 10 August 2015
Digestion products were loaded on 1% agarose gel to purify.
Sample:
20 µL DNA (bslA).
4 µL 6x buffer.
Ladder:
2 µL Thermo Scientific GeneRuler 1kb DNA ladder, ready to use.
Gel was run on 100V for 30 minutes. Gel results show a band in sample 1 of 600bp and in sample 2 a band of 2000bp. This corresponds to the size of bslA and pSB1C3. Gel was cut and dissolved in 2:1 binding buffer of the PCR purification kit and purified according to protocol. Samples were stored as: bslA dig (10,1ng/µL, Nanodrop value) and: pSB1C3 dig (6,2ng/µL, Nanodrop value).
20:15, 31 August 2015 - 22:30, 10 August 2015
bslA dig was ligated into pSB1C3 dig. Ligated at room temperature for 2 hours.
Component
Amount
bslA DNA
10 µL
pSB1C3 DNA
10 µL
T4 ligase
0.5 µL
T4 ligase buffer
3 µL
\( \mathrm{H_2O}\)
6.5 µL
Components used for ligation.
22:30, 31 August 2015 - 00:30, 1 September 2015
Ligation product (30µL) was transformed into 10µL NEB DH5 alpha competent cells. Cells were plated on LB + chloamphenicol agar plates.
17:00, 1 September 2015 - 21:00, 1 September 2015
Transformation was checked. On the plate were 10 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
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 ten colonies have the correct insert size.
21:30, 1 September 2015 - 10:00, 2 September 2015
One colony was grown overnight in liquid LB + chloramphenicol at 37°C in a shaker incubator at 200rpm.
10:30, 2 September 2015 - 11:00, 2 September 2015
A 50% glycerol stock was made from 500µL culture and stored at -80°C.
11:00, 2 September 2015 - 12:00, 2 September 2015
2,5mL of culture was miniprepped and stored at -20°C as: bslA (16,7ng/µL, Nanodrop value). Sample was sent in for sequencing and has the correct suffix.