MIME-Version: 1.0 Content-Location: file:///C:/2359D654/TransProt.htm Content-Transfer-Encoding: quoted-printable Content-Type: text/html; charset="us-ascii" Transgenesis Protocols

Transgenesis Protocols

 

Construction of Micropipettes

 

  1. Fine glass tubes (Microcaps, Dramont) are cut into two pi= eces by applying heat in a puller (PB-7, Marishige). The front parts of the micropipettes are slightly broken by fine forceps to ensure the outlets measuring the tips as around 40um
  2. The tips are checked under microscope whether the prepared pipettes have the same shapes and measurements.

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Siliconization of Micropipettes, Capillary Tubes and 1.5 ul Tubes

 

  1. By using special dropper and pipette, the interior walls = of the micropipettes, capillary tubes and 1.5ml tubes are siliconized with Si= gmacoat (Sigma).
  2. The siliconized apparatuses/items are then air dried and checked under microscope.
  3. If there is any liquid left inside the siliconized micropipettes, the liquid is removed by a special air-sucking machine.=

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.

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Purification of DNA

 

To pur= ify, the linearized DNA is extracted with Phenol Chloroform Isopropanol (PCI).

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1.      150ul of TE (1mM TE, PH 8.0) and 250ul of PCI are added with 100ul digested DNA mixture and vortexed and centrifuged at 15000 rpm for 3-5 minutes at 4C.

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.<= /o:p>

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.

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Electrophoresis

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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.

 

Preparing DNA Solution

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Approp= riate amount of TE is added to the DNA to adjust the concentration of the DNA to 0.1ug/ul.

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Preparation of Female Frogs

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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

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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

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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.

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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).

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