0.5 µl Expand High Fidelity Polymerase (1) (Roche)
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50 µl Final volume
PCR program:
1x:
94ºC 2´
10x:
94ºC 15´
Annealing Temp 30´´
72ºC 1´ (≤ 1kb) or 1.30´´(> 1kb)
20x:
94ºC 0.15´´
Annealing Temp 30´´
72ºC 1´ (≤ 1kb) or 1.30´´(> 1kb) + 10´´/cycle
1x:
72ºC 7´
∞:
15ºC
A2) Gel extraction:
Separate the PCR product on a 1% agarose gel.
Use a Gel Extraction Kit to purify the PCR band from the gel.
Run 1µl in a 1% agarose gel in order to estimate the concentration compared to a reference sample, e.g. phage-λ.
A3) Cloning in a GATEWAY TOPO (Invitrogen):
In a 0.5ml tube, mix:
50-100 ng (4 µl max.)- fresh PCR product (do not freeze for a long period)
1 µl - entry vector diluted 1/5
1 µl - saline solution buffer
µl - mQH2O
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6 µl Final volume
Leave 5 minutes at room temperature.
Place on ice and proceed with the transformation.
A4) Transformation:
Mix 3 µl of DNA+vector with 50-100 µl of competent cells (DH5α , TOP10 or Match1).
Incubate on ice 5 minutes minimum.
Plate all the volume in a 1% LB-agar plate with spectinomicine (100 mg/l).
Pre-warm the plates at 37ºC.
Leave the plates overnight at 37ºC.
A5) Confirming positive colonies:
Pick up 3 colonies with a yellow tip and inoculate them into 15ml non-hermetic tubes with 2ml of LB with spectinomicine (100 mg/l).
Incubate overnight at 37ºC in a shaker.
Purify the DNA with a Plasmid Miniprep Kit.
Resuspend in 40 µl of elution buffer.
Digest 3 µl with the EcoRI restrictase in a final volume of 20 µl and run the digestion in a 1% agarose gel to check whether the vector contains the insert.
A6) Recombination with the destination vector:
Mix in a PCR eppendorf:
1 µl from a 1/5 dilution of the TOPO containing your insert (aprox.100ng)
1 µl from a 1/4 dilution of the destination ZED vector (100 ng)
0.5 µl of the LR clonase enzyme (Invitrogen).
Leave it minimum 1h at 25ºC in the thermocycler.
Add 0.25 µl of Proteinase K (2 µg/µl).
Incubate 10 minutes at 37ºC.
Place on ice and proceed with the transformation.
A7) Transformation:
Mix 1.25 µl of the reaction with 50 µl of Match1 competent cells (Invitrogen).
Place on ice 20 minutes minimum.
Heat-shock 45 seconds at 42ºC.
Place on ice 2 minutes.
Add 1ml SOC medium and leave 1h at 37ºC.
Pre-warm the plate at 37ºC.
Spin 5 minutes at 2000rpm, remove 800 µl and resuspend the pellet in the remaining 200 µl.
Plate all in a 1% LB-agar with ampiciline (100mg/l).
Leave the plates overnight at 37ºC.
A8) Confirming positive colonies:
Pick up 2 colonies with a yellow tip and inoculate them into 15ml non-hermetic tubes with 2ml of LB with ampiciline (100 mg/l).
Incubate overnight at 37ºC in a shaker.
Purify the DNA with a Plasmid Miniprep Kit.
Resuspend in 60 µl of elution buffer in a safelock 1.5ml tube.
Set up a confirmation PCR with the primers used to amplify the insert.
PCR set up:
1 µl DNA
2 µl Buffer 10X
2 µl dNTPs 2mM
1.5 µl Primers (Fwd+Rev mix at 10µM)
0.25 µl Taq polymerase
13.25 µl mQH2O
-----
20 µl Final volume
PCR program:
1x:
95ºC 2´
30x:
94ºC 15´
55ºC 30´´
72ºC 1´
1x:
72ºC 7´
∞:
4ºC
A9) Phenol/Chloroform extraction:
Add 20µl 3M NaAc + 120µl H20.
Add 1 Vol. Phenol/Chloroform.
Centrifuge 5 minutes at 13000rpm.
Transfer upper phase to a new 1.5ml safelock tube.
Add 1Vol. Chloroform/IAA (24/1).
Centrifuge 5 minutes at 13.000rpm.
Transfer upper phase to a new 1.5ml safelock tube.
Add 2 Vol. EtOH 100% and leave at least 1h at -20C.
Spin 15 minutes at 13.000rpm.
Wash with EtOH 70%.
Spin 15 minutes at 13.000rpm.
Remove EtOH and air dry.
Resuspend in 10µl mQH2O.
Measure the concentration by spectrophotometry (Nanodrop).
Make a dilution at 50 ng/µl.
B) The Enhancer Screen (ES)
Figure 1. The Enhancer Screen (ES)
B1) Injection:
In a 0.5ml tube, mix:
1 µl construct DNA [50 ng/µl]
1 µl transposase mRNA [50 ng/µl]
0.2 µl red phenol
Load a capillary of 1mm of diameter with the previous mix. The capillary is pulled-out in a horizontal puller.
Inject with a micro-injector 5-10 nl of the mix into one-cell stage zebrafish embryos.
Keep the embryos at 28ºC in embryo medium. Change the medium twice a day.
Select embryos with a broad RFP expression (transgenesis control, see Fig. 2) and raise them to adulthood.
B2) F0 screening:
F0 adult fishes were out-crossed with wild-type zebrafish. Embryos from these crosses were screened for GFP expression.
Each enhancer expression pattern was validated through the identification of 3 founders with the same GFP expression pattern.
B3) Stable transgenic ES lines establishment:
F1 embryos with positive GFP expression patterns were selected to establish stable transgenic lines by growing F1 embryos to adults.
C) Vectors used in the Enhancer Screen
Zebrafish Enhancer Detector (ZED) vector (1. Bessa et al.)
The ZED vector (Fig. 2A) consists of:
GFP under the control of a minimal promoter;
Insulator sequences to shield from positional effects;
Positive control for transgenesis (cardiac actin RFP).
Figure 2. (A) Scheme of the ZED vector. (B) Enhancer expression (GFP) in a F0 injected zebrafish embryo. Control cardiac actin expression is shown in red (RFP).
References:
1. Zebrafish Enhancer Detection (ZED) Vector: A New Tool to Facilitate Transgenesis and the Functional Analysis of cis-Regulatory Regions in Zebrafish.
