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Sequence Analysis of Single Polymorphism in the Factor V Gene - page 6 / 15

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Biol  420                                                                                                                                          Fall 2007

a.

Carefully transfer 25 ul of each PCR sample to a properly labeled eppendorf tube and add 5 ul of loading dye to each.  Note:  Be sure to take the sample from beneath the mineral oil layer.

b.

To prepare you genomic DNA samples, transfer 2 ul of your original genomic DNA sample to a properly labeled microfuge tube.  Add 8 ul of H2O and 2 ul of loading dye to each.

c.

Mix samples well and centrifuge for 10 seconds to ensure that your sample has collected at the bottom of the tube.

2. Once your agarose gel has solidified (for confirmation of this, you may ask your instructor), place it in an electrophoresis chamber and add enough 1x TAE to cover the gel.

3. Carefully remove the comb to reveal the wells.  Note:  Be careful not to disrupt the wells when removing the comb.

4. Load samples as follow:

d.

Genomic DNA 1 (12 ul)

e.

PCR sample 1 (30 ul)

f.

Genomic DNA 2 (12 ul)

g.

PCR sample 2 (30 ul)

h.

100 bp DNA marker (10 ul)

5. Run the gel for 1 hour at 90 volts

6.  Visualize the DNA products on your gel under UV light. Take a photo of your gel.   Note:  Always wear glasses when looking at the UV light.  It can burn your eyes, which is very painful.

-PCR product should be between 185-205 bp in size.

Collecting the DNA fragment for Sequencing.

1.

After taking a photo of your gel, you will use a scalpel to cut out the band of interest.  You should cut out the band with the agarose gel exposed to the UV light, and then turn out the UV.  Removal of the band can be completed with the UV light off.  This will limit your exposure to the UV light and prevent possible mutations to the PCR product in your agarose gel.

2.

Prepare a GenElute Column.  Label the column appropriately.  Add 100 ul TE to each Gen Elute Column and spin at 14000 rpm for 10 sec to equilibrate the column.

3.

Transfer the column to a properly labeled microfuge collection tube.

4.

Pplace each piece of agarose containing a DNA fragment to a prepared GenElute Column.

5.

Centrifuge the spin column at maximum speed for 10 minutes.  The purified DNA  is now located in the collection tube.  So you can discard the column.

Purification of GenElute DNA by Ethanol Precipitation

1.  To the recovered GenElute DNA add 2.5 volumes of 95% Ethanol and 0.1 volumes

of 3 M sodium acetate.

2.

Mix well by inverting the tube several times and incubate samples at -20oC

overnight.

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