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Sequence Analysis of Single Polymorphism in the Factor V Gene - page 7 / 15

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Biol  420                                                                                                                                          Fall 2007

Lab 1.4:  Purification of DNA fragments and Preparing Your Sequencing Reactions.

1.

Taking the sample from the previous day, centrifuge for 15 minutes at maximum speed

2.

Carefully discard the supernatant.  Be sure to retain the pellet in your tube.

3.

Add 250 ul of 70% ethanol.  Then, flick the tube to ensure that your pellet is dislodged and washed well by the alcohol.  

4.

Resuspend the DNA pellet in 30 ul of water or TE buffer.  Heat at 50 oC for 15 minutes and store at 4 oC until ready to use in your sequencing reaction.  For longer storage, place you purified DNA at -20 oC.

Preparing your Sequencing Reaction:

1.

Determine the concentration of your DNA by taking an OD260 reading in the spectrophotometer in 218.  Prepare your samples in 1.5 ml microfuge tubes as follows

a.

Blank = 1ml dH20

b.

Sample 1 = 5 ul sample 1 + 955 ul H20

c.

Sample 2 = 5 ul sample 2 + 955 ul H20

d.

Sample 3 =5 ul sample 3 + 955 ul H20

Turn on the spectrophotometer.  Transfer the blank sample to a cuvette, place in the spectrophotometer and select “blank”.  Then place your sample 1 in a cuvette and select   “sample read “ and record the absorbance reading.  Repeat this step for each sample.

Calculate the concentration of each sample as follows:

OD260 x 50 x 200 (dil. Factor) =  ___ng/ul

2.

Prepare the template/primer mix for your sequencing reactions by adding the following in the order listed to a 1.5 ml microfuge tube:

___ul (100 fmoles) of the DNA

1.5 ul of IRDye 700 Fwd Primer (1.0 pmol/ul)

1.5 ul IRdye 800 Rev Primer (1.0 pmol/ul)

2.0 ul of Thermo Sequenase Reaction Buffer

1.0 ul of 2.5 mM dNTP nucleotide mix

2.0 ul of Thermo Sequenase DNA Polymerase,

___ ul of ddH2O for a final volume of 17.0 ul.

Note: The Fwd and Rev primers are the M13 forward and reverse sequences shown below labeled with either a IRDye800 and IRDye 700, respectively.

M13 Forward:  5’-CACGACGTTGTAAAACGAC-3’

M13 Reverse:  5’-GGATAACAATTTCACACAGG-3”

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