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Sequence Analysis of Single Polymorphism in the Factor V Gene - page 8 / 15

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Biol  420                                                                                                                                          Fall 2007

2.

Mix well by pipeting.

3.

For each template/primer mix, label a set of four 0.2 ml PCR tubes A,T,C, and G and add 4.0 ul of the A reagent to the A tube, the T reagent to the T tube, etc.

4.

Add 4 ul of the template /primer mix to each of the A,T,C, and G reagent tubes.

5.

Add 20 ul of mineral oil down the side of the tube.

6.

Place into the thermal cycler and cycle as follows:

1 cycle of :  (File 24)

92oC – 2 minutes

30 cycles of:  (File 25 – machine 2)

92oC – 30 seconds

50oC – 30 seconds

70oC – 1 minute

4oC – Hold (file 8 – machine 2)

Select File 24 and hit run to start the machine.

Lab 1.5:  Loading Sequencing Gel

Preparing the Samples

1.

Once cycling is completed remove the PCR tubes.

2.

To each tube add 4.0 ul of IR2 stop solution below the oil.

3.

Using a pipetter remove all liquid from beneath the oil in each PCR tube, it will be purple in color, that is not the mineral oil and dispense onto a sheet of parafilm. Make sure to keep the liquid from each tube separate.  Once there are 4 drops corresponding to A, T, C, and G slowly tilt the parafilm causing the drops to slowly move down the parafilm.  This will get rid of any excess oil.

4.

Remove the drops from the parafilm using a pipetter and place into new properly labeled PCR tubes.

Preparing the Sequencing Gel

1.

Carefully wash and dry plates.

2.

Place plates flat on the bench top (be sure that they are beveled side up) and spray the plates 100 % Isopropanol.  Then use a paper towel to dry to plates.

3.

Prepare Bind Silane Solution by adding 25 ul of Bind Silane Stock solution  and 25 ul of 10% acetic acid to a microfuge tube.  Then pipette 25 ul to each plate (in the region where the comb will be located) and use a cotton swab to spread the solution across the width of the plate in that area.

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