M13 forward and m13 reverse primers

One-cell stage zebrafish embryos were injected with ∼2-3 nl of a solution containing 250 ng/µl Cas9 mRNA, 15 ng/µl sgRNA and 5-50 ng/µl template DNA.
For PCR amplification of the repaired alb locus from F₁ larvae, the tissue samples were incubated in TE supplemented with 5% Chelex-100 (BioRad) and 10 µg/ml Proteinase K (Roche) for 4 h at 55°C and 10 min at 95°C and then stored at 4°C. 1 µl of the supernatant was used as template in a standard 25 µl PCR with primers 1097 and 1098. The PCR product was cloned into pGEM-T Easy and sequenced with M13 forward and M13 reverse primers (Invitrogen).

DNA was extracted from 13 individual colonies using the KAPA Express Extract Kit (KAPA Biosystems), with slight modifications of the reaction volume: the total volume was 20 μL, consisting of 2 μL Express Extract Buffer, 0.4 μL Express Extract Enzyme, and 17.6 μL dH₂O. Lysis incubation was done at 75°C for 20 min, followed by an enzyme inactivation step at 95°C for 5 min. The 16S rRNA genes were amplified by PCR using the universal bacterial primers 27 forward and 1492 reverse [64 ]. The 18S rRNA genes were amplified using the universal eukaryotic primers 82 forward [65 (link)] and Medlin B reverse [66 (link)].
The obtained PCR products from each colony were cloned separately using the TOPO-TA cloning kit (Invitrogen) according to the manufacturer’s instructions. For screening of the 16S rRNA and 18S rRNA genes, 10 to 15 clones each were picked and controlled for the correct size by PCR with the M13 forward and M13 reverse primers (Invitrogen). Polymerase chain reaction products of the correct size for the 16S rRNA gene (~1,500 nt) and for the 18S rRNA gene (~1,800 nt) were fully sequenced via Sanger sequencing and further analyzed using the program CodonCode Aligner (CodonCode Corporation; www.codoncode.com).