Genome Engineering by Transgene- Instructed Gene Conversion in C. elegans - Caenorhabditis Elegans: Molecular Genetics and Development - page 77

sequence and in order to minimize it, PCR conditions need to be set up for each
MosTIC experiment.
(b) PCR Conditions Setup
We observed that jumping PCR could be minimized by reducing the annealing
time, increasing the annealing temperature, and/or diluting 10-100 times the
worm lysates before starting PCR.
To optimize the PCR conditions and minimize ''jumping PCR,'' proceed as
follow:
1. Construct a positive control for your PCR ( Fig. 3B and 3C ). It must contain the
full-length repair template and an additional 200-300 nucleotides long geno-
mic fragment including the primers used for MosTIC-engineered allele
screening. Using the Phusion high-fidelity DNA polymerase (Finnzymes) or
any other high-fidelity polymerase, amplify (i) the full-length repair template
on the previously constructed plasmid using P1 and P4 for KO/del repair
template and P1 0 and P6 0 for KI repair template and (ii) the 200-300 neuclotide
long genomic fragment adjacent to the short arm of the repair template. This
latter is amplified with primers P9-P10 and P11 0 -P12 0 respectively. P9 and
P11 0 contain in their 5 0 end a tail of 24 nucleotides that overlaps with the end of
the short arm of the repair template and fusion PCR is performed using
P1-P10 and P1 0 -P12 0 , respectively. The PCR product is subcloned using
the Zero Blunt PCR cloning kit (Invitrogen) or any other cloning kit.
2. For each transgenic line, pick three to five non-heat-shocked transgenic
animals on a 6 cm fresh NGM plate. Grow these worms and their progeny
for 1 week at 20 C. Collect the mixed nontransgenic/transgenic worm pop-
ulation with 1 mL of M9 1 buffer and transfer them to a 1.5 mL tube. Place
on ice for 10 min for sedimentation. Collect 50 m L at the bottom of the tube
and transfer to 0.2 mL PCR tubes. Add 50-100 m L of worm lysis buffer
complemented with proteinase K. Perform lysis at 65 C for 2-3 h and
inactivate proteinase K by incubating the lysate at 95 C for 20 min. Such
lysates can be stored at -80 C for at least 3 weeks.
3. To set up PCR conditions, use 1 m L of each lysate, a 1 ng/ m L and a 10 -2 ng/
m L dilution of the control plasmid preparation. For each sample, perform the
following PCR program (using a standard Taq DNA polymerase):
- step 1: 3 min at 95 C,
- step 2: 30 s at 95 C,
- step 3: 15 s at annealing temperature,
- step 4: 2 min at 72 C temperature,
- step 5: cycle 29 times from steps 2 to 4
- step 6: 5 min at 72 C.
Make a gradient of annealing temperature ranging from 55 to 70 C. For the
nested PCR, dilute the first PCR 100 times and perform the second PCR at the
same annealing temperature.