Synbio1

1. Synthetic Biology 1: Copying DNA
General:
The participants learn the basic techniques of working in molecular biology.
Understand the principle of a PCR and can detect DNA using gel electrophoresis.
Setting:
A patient was diagnosed with a multidrug-resistant bacterial infection. No antibiotic is
effective. We isolate and cultivate the pathogen from a patient sample for further
therapeutic approaches. Candidates are then identified for an alternative treatment
with bacteriophages. (Review Microbio for beginners).
Now it should be checked which resistance gene the germ contains. For this we
have already extracted the bacterial DNA and identified possible genes.
Overview:
Duratio
n
What ? Content and goal Material
60 min lecture “Introduction to
molecular biology”
● DNA the code of life
● PCR theory
● Gel electrophoresis
❏ Introductory
60 min PCR: How to copy DNA ● PCR basics ❏ ddH2O
❏ R primer
❏ F primer
❏ DNA template
❏ mix
80 min gel electrophoresis ● Gel for gel electrophoresis is
poured
● The gel is loaded and
energized
❏ H20 dist.
❏ agarose tablets
❏ Gel chamber
❏ loading buffer
❏ 100 Bp ladder
❏ TAE buffer
30 min “Evaluation and error
analysis”
● The separated DNA is
analyzed and possible
sources of error are
discussed

2. Lecture “Introduction to molecular biology”
Objective:
Introduction to the participants.
Material:
● Presentation
Execution:
The presentation is given and the questions of the participants are answered.
PCR: How to copy DNA
Aim:
The theory is put into practice. The participants learn how to pipette small volumes and learn
how to handle DNA sensitively
. Material:
❏ F
❏ mix ddH2O
❏ primer
❏ R primer
❏ DNA template
Implementation:
It is divided into 2 groups each group shows different genes. An Eppi for each rehearsal.
One possibility is shown here:
Control
❏ 5uL master
❏ mix 1uL DNA
template
❏ 14 uL ddH2O
IDT pucD (purchased primers)
❏ 5uL master
❏ mix 1uL DNA template
❏ 14 uL ddH2O
❏ 2uL F primer
❏ 2uL R primer
pucdt (self-printed)
❏ 5uL master
❏ mix 1uL DNA
template
❏ 14 uL ddH2O
❏ 2uL F-Primer
❏ 2uL R-Primer
Theory:
For a sample with a volume of 25 uL the following concentrations are required:
● 5uL master
● mix 10ng/ul DNA(purified) or 100ng/ul(extraction)
● 200 nmol/uL primer

3. https:/
/international.neb.com/proto
cols/0001/01/01/taq-dna-poly
merase-with-standard-taq-bu
ffer-m0273
STEP
TEMP TIME
Initial Denaturation 95°C 30 seconds
30 Cycles 95°C
45-68°C
68°C
15-30 seconds
15-60 seconds
1 minute/kb
Final Extension 68°C 5 minutes
Hold 4-10°C
gel electrophoresis
Objective:
theory is applied in practice. The participants learn the basic technique of DNA separation
and understand the principle
Material:
❏ H20 dest.
❏ agarose tablets
❏ Heating stirrer
❏ Gel chamber
❏ loading buffer
❏ DNA samples
❏ 100 bp ladder
❏ TAE buffer
Procedure:
1 gel is prepared. For this purpose, an agarose tablet is dissolved in 50mL dist. H20 in a
150mL Erlenmeyer flask. dissolved and boiled with a heating stirrer. The solution is cooled to
around 50 degrees and then transferred to a gel chamber. The slot comb is inserted and the
whole thing is cooled down. Cover the cooled gel with TAE buffer.
Each TN receives a sample to fill a slot.
To fill the slots:

4. ● It is important to ensure good lighting conditions.
● Each sample (10 uL) receives 2 uL loading buffer
● The slots on the edge are to be
. Then attach the gel. 10 minutes at 50 volts, then let the gel run for about 20-30 minutes at
90 volts.
Evaluation and error analysis
Aim:
The participants see the copied DNA and learn how to determine the length of a DNA
fragment using the DNA ladder
Material:
● Gel from gel electrophoresis
Implementation:
The gel is photographed on a UV screen and then shown to everyone on the screen and
examined.