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Sequence Analysis of Single Polymorphism in the Factor V Gene - page 5 / 15

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Biol  420                                                                                                                                          Fall 2007

e.

81.6 ul Ultrapure ddH2O

Primers designed for this experiment:

5’-CACGACGTTGTAAAACGACTAATCTGTAAGAGCAGATCC-3’

       M13 Forward SeqFactor V Forward Seq.

5’-CCATAACATTTCACACAGGTGTTATCACACTGGTGCTAA-3’

       M13 Forward SeqFactor V Forward Seq.

2.

This should be a total of 100 ul

3.

Overly the PCR reaction with a drop of mineral oil.  This will prevent the evaporation of your sample

4.

Place the PCR samples in the thermal cycler and set the machine to perform the following

1 cycle of:  (File 19)

94o C - 5 minute

30 cycles of: (File 18)

94o  C - 1 min

50 o C - 1 min

72o  C  - 1 min 30 sec.

Overnight hold at 4 o C  (File 8)

*Enter File 19 and select run.

Lab 1.3:  Analysis of PCR Reaction and Isolation of DNA Fragments

Preparation of Agarose Gel

1.

Measure 2 grams of PCR or low melt agarose and carefully add it to a 200 ml flask.

2.

Prepare 1 liter of 1 X TAE (Measure 20 ml of 50X TAE and add 980 ml of ddH2O.  Mix well.)

3.

Then add 100 ml of 1X TAE to the flask containing the agarose powder.  Place a paper towel in the top of the flask to absorb moisture.

4.

Gently swirl to mix and microwave to melt agarose.

5.

Allow agarose to cool for 10 minutes and then ask your instructor to add 5 ul of EtBr to melted agarose.

6.

Place gel tray into a casting apparatus and pour melted agarose into sealed tray and let solidify.  This should take about 20 minutes.

7.

While your agarose gel is solidifying, you can prepare you PCR samples (see below)

Preparing your PCR and Genomic DNA Samples and Loading the Gel

1. Prepare your PCR samples for the gel:

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