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Sequence Analysis of Single Polymorphism in the Factor V Gene - page 4 / 15

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Biol  420                                                                                                                                          Fall 2007

Experimental Design:

Lab 1.1:  Extract DNA from Buccal (cheek) Cells

(as in Manual ArchivePure DNA Purication by 5 Prime)

Sample Collection and Cell Lysis

1.

Put 300 ul of lysis solution into 1.5 ml microfuge tube.

2.

Brush inside of check with at least 10 strokes with a sterile brush

3.

Insert into the 1.5 ml microfuge tube containing the 300 ul of lysis solution

4.

Swish around for approximately 1 minute

5.

Cut off tip with sterile blade or scrissors and leave in the 1.5 ul microfuge tube and label microfuge tube(s)

6.

Add 1.5 ul of Proteinase K solution (20 mg/ml) to cell lysate, mix by inverting 25 times and incubate at 55 ۫ C for 1 hour.

7.

After the hour has elapsed remove brush making sure to scrape the brush along side of the tube to remove any excess lysate from the collection brush head

8.

Add 1.5 ul of RNase A solution to the tube, invert 25 times, and incubate for 20 minutes at 37 ۫C, once the 20 minutes has elapsed place in an ice bath for 1 minute to bring the sample back down to room temperature.

9.

Add 100 ul of Protein Precipitation Solution  to the cell lysate

10.

Vortex at high speed for 20 seconds to mix the Protein Precipitation Solution uniformly with the cell lysate.

11.

Place tube in ice bath for 5 minutes

12.

After the 5 minutes centrifuge at 14000 RPM for 3 minutes.  Make sure the proteins have formed a tight, white pellet.  If it is not tight repeat steps 10,11 and 12.

13.

Pour the supernatant containing the DNA into a clean 1.5 ml microfuge tube containing  300 ul 100% Isopropanol and 0.5ul (20 mg/ml stock) Glycogen Solution.

14.

Invert 50 times, incubate at room temperature for 5 minutes

15.

Once the 5 minutes are completed centrifuge at 14000 RPM for 5 minutes.  DNA may be visible as a small white pellet.

16.

Pour off supernatant and drain tube on clean absorbent paper.  Add 300 ul of 70% Ethanol and invert tube several times to wash DNA.

17.

Centrifuge at 14000 RPM for 1 minute.  Carefully  pour off the ethanol and allow to dry for at least 15 minutes

18.

Add 20 ul of DNA Hydration Solution overnight at room temperature then store at -4 ۫ C

Lab 1.2:  Factor V PCR

1.

Start with 2 ul of genomic DNA (then add)

a.

10 ul 10X PCR buffer (+MgCl2)

b.

2 ul (200uM) dNTPS (10 mM)

c.

1.9 ul (0.3 uM) forward primer

d.

1.5 ul (0.3 uM) reverse primer

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