Creating zebrafish transgenics:
1. Choose appropriate oligos to amplify genomic region of interest. My advice on oligo length is to
range between 25 and 30 nucleotides with 50% G/C. Make sure you have fresh (less than 1 month)
Spectinomycin plates for step 7.
2. Perform PCR reaction as below using Phusion. Choose an annealing temp that is 5C lower than
oligo Tm for first reaction. Set machine in gradient mode if you need many different temps.
Reaction:
5 ul
5 x HF buffer (phusion hi fidelity buffer)
0.5 ul
dNTPs (10 mM-part of Phusion kit)
0.75 ul
F primer (20 uM concentration)
0.75 ul
R primer (20 uM concentration)
0.5 ul
genomic DNA (100-500 ng total)
0.25 ul
Phusion HS (hot start version of Phusion NEB)
xxx ul
dH2O
25 ul
VT
Perform PCR reaction according to Phusion directions
1 min 98C
:20 sec 98C
:20 sec 53C
40 cycles
1:00 min 72C
10 min 72 C
4 C hold
3. Run out PCR reactions on appropriate agarose gel. I use 1.1% agarose/1X TAE gels with 1 ul ETBr
in gel.
4. Isolate bands from your reactions. Use Qiagen band purification kit according to instructions.
5. Perform Taq 5’ A addition reaction.
Reaction:
16 ul
your purified PCR product
2 ul
10 X Ex-Taq buffer
1 ul
2.5 mM dNTPs (from Ex Taq kit)
1 ul
Pickard Taq (do not use proofreading mix!)
20 ul
VT
Incubate reaction in PCR machine at 72C for 10-15 min
6. Immediately use this to set up TOPO cloning reaction:
Reaction:
4.5 ul
1 ul
0.5 ul
your A-tailed PCR product from above
Salt solution
pCR-8GW TOPO vector
Incubate reaction at RT for 5 min and immediately place on ice, transform as below
7. Tranform 3 ul of your TOPO cloning reaction into 25 ul of TOP10 cells. Incubate on ice 30 min.
Incubate at 42C for :30 sec. Place immediately on ice 2 min. Add 125 ul SOC and incubate 37C 30
min. Plate all of transformation on LB-spectinomycin plate (*** pCR TOPO 8GW is spectinomycin
resistant not Kan/Carb/Chlor).
8. Grow up two minipreps from each cloning reaction and digest product with EcoRI. Pick ones that
have inserts (most will).
9. Linearize pXIG-GW with EcoRI and purify band with Qiagen kit. Can store extra at -20C at
concentration of 150 ug/ul.
10. Perform LR clonase reaction
1 ul miniprep (approximately 200 ng)
2 ul of pXIG GW at conc of 150 ug/ul (approximately 300 ng)
2 ul of 5x LR clonase buffer
2 ul of LR clonase mix
3 ul of TE
10 ul Vt
Incubate at 25 C for 16 hours.
***Treat with xx ul Proteinase K 37C 10 min
Can store at -20C.
11. Transform 5 ul of LR clonase reaction into 25 ul Omnimax cells
12. miniprep 4 colonies of each and cut with BamHI and XhoI (Buffer C). Your vector should have a
fragment that is 200 nucleotides bigger than your insert. If it has a 2.2 kb fragment, that is the starting
vector. To be sure run control digest with the starting plasmid pXIG-GW.
13. maxiprep your plasmid and pCS2-TP. Linearize pCS2-TP with NotI and synthesize mRNA with
SP6 based mMessageMachine kit (Ambion). Coinject your plasmid and TP mRNA at 1-2 cell stage.
Note: both initial TOPO and LR clonase reactions are 0.5x reaction conditions. There should be a vast
excess of colonies. It is likely that .25x reactions would also be successful.