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Transgenesis Protocols
Construction
of Micropipettes
Siliconization
of Micropipettes, Capillary Tubes and 1.5 ul Tubes
Digestion
of Plasmid DNA
1. The plasmid DNA is digest= ed with a suitable restriction enzyme (e.g. Hind III, Sa II etc.) with a proper combination of buffer (1/10th of total volume) and water to make= it linearized. In case of 1ug/ul DNA, 1-5 ul of DNA, 20-40 units of enzyme and the rest of the volume is filled with autoclaved water.
2. The mixture is then incub= ated at an ambient temperature (usually 37 C) for 2-3 hours.
3. To check and compare, the= digested DNA and the non-digested circular plasmid DNA are run in 1% agarose gel along= with dye of the 1/6th volume of the sample DNA.
4. After electrophoresis, th= e gel is stained with Mupid Blue dye for 1.5 minutes and then washed in 70% Et-OH for= two times, 1minute each followed by washing in water for several times, 1minute each.
5. Then the DNAs in the gel = are observed on a light box and photographs are taken.
Purification of DNA
To pur= ify, the linearized DNA is extracted with Phenol Chloroform Isopropanol (PCI).
1.
150ul of TE (1mM TE, P
2.&n=
bsp;
The upper layer is
collected in a new tube and PCI of the same volume of the sample is added f=
ollowed
by vortex and centrifuge at the same speed and duration.
3.&n=
bsp;
Again the upper layer=
is
collected and 250ul of chloroform is added to remove the phenol, vortexed a=
nd
centrifuged.
4.&n=
bsp;
The lower layers of every centrifuge can also be collected and
treated in the same manner.
5.&n=
bsp;
Now 250ul supernatant=
is
collected from the treated upper layers and if possible, 150ul from the tre=
ated
lower layers (Total volume of extracted DNA, 250+150=3D400ul).
6.&n=
bsp;
T=
hen 40ul sodium
acetate (1/10th of the volume of the extracted DNA) along with 4=
00ul
of isopropanol (same vol of the sample DNA) are added to make the grand tot=
al
as 400ul+40ul+400ul=3D840ul.
7.&n=
bsp;
It is kept in freezer=
(-20oC)
for 20 minutes and then spinned down at 15000 rpm for 15 minutes.
8.&n=
bsp;
The supernatant is re=
moved
and same volume of 70% Et-OH is added to it to remove the sodium acetate sa=
lt
and centrifuged at same rpm for five minutes.
9. The supernatant is remove= d out and allowed to dry for 5-10 minutes.
10. Then the DNA is dissolved= in 10ul TE.
Electrophoresis
To kno= w the concentration of the DNA, 1 ul of this DNA solution is diluted with 99ul of= TE (1/100 th of the vol) and measured at 260 A= o and the value is multiplied by 5 to express the amount in unit as ug/ul. = span>
Preparing DNA Solution
Approp= riate amount of TE is added to the DNA to adjust the concentration of the DNA to 0.1ug/ul.
Preparation of Female Frogs
1. Female frog is pre-primed= by injecting 100-200ul (50-100 units with a concentration of 0.5 unit/ul 0.6% NaCl) of PMSG (Serotropin) 2-14 days before priming.
2. Then the frog is primed o= n the previous day of egg-collection by injecting 400 units (1unit/ul) of HCG (Gonadotropin) and is kept in 1xMMR at 16-18˚C.
Preparation of Eggs
1. 35 mm petridishes are fil= led half with agar or agarose, dried and kept at 16˚C with 6% ficol in 0.4= xMMR.
2. Unfertilized eggs are dej= ellied with 2% cysteine in 1xMMR.
3. Then the eggs are washed = with cold 1xMMR and put in the petridishes.
Items for Microinjection
1. Sperm nuclei (100nuclei/n= l)
2. 100mM MgCl2
3. Plasmid DNA solution (Gene construct with EGFP).
4. Heated egg extract (HEE)<= /span>
5. Sperm dilution buffer (SD= B)
6. Unfertilized dejellied eg= gs
7. 0.1xMMR
Preparation of Microinjecting Materials
1.&n= bsp; 3ul of DNA solution + 1ul of 100mM MgCl2 = + 1ul of sperm nuclei are mixed and kept on ice for 5 minutes.
2.&n= bsp; 5ul of HEE is added t= o it and is kept at room temperature for 5 minutes.
3.&n= bsp; Then it is diluted wi= th 290ul of SDB, so now the total volume is 300ul.
Microinjection
1. A siliconized capillary t= ube is set to the yellow tips of pipetteman and 30ul of the solution is sucked in = it and then it is passed into the siliconized micropipette and first the tube = is detached from the pipette and then the micropipette is detached from the tub= e.
2. The eggs are injected at = or around the animal pole (anterior side) and the injected number of eggs and = the injected time is recorded.
3. The dish of the injected = eggs is kept at 16˚C for 2 hrs and observed under a microscope.
4. The fertilized eggs are selected a= nd put in a new dish with 0.1xMMR containing 10ug/ml of antibiotic (Gentamicin Rea= gent Solution, Invitrogen).