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Sequence Analysis of Single Polymorphism in the Factor V Gene - page 10 / 15

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Biol  420                                                                                                                                          Fall 2007

6.

Slide the upper buffer tank into place.  Be careful not to let the gasket touch or drag against the gas plates during installation as this may displace the gasket.  Then tighten the upper clamp knobs “finger tight”.  

7.

Open the 4300 instument door and place the lower buffer tank into position at the base of the heater plate.  

8.

Then mount the gel apparatus on the instrument against the heater plate with the bottom of the apparatus inside the lower buffer tank.  Check to see that the support arms are holding the gel assembly on the instrument are seated evenly on the instrument.

9.

Fill the buffer tanks as specified with 0.8 X TBE.  Prepare 0.8X TBE by adding 80 ml of 10 X TBE to 920 ml of ultrapure dH2O.  Fill the upper buffer tank to the Max Fill Line and then pour the remaining O.8 X TBE into the lower buffer tank.

10.

Fill a 20cc syringe with buffer from the upper tank, add the 22 guage needle and flush the wells with buffer to remove crystallized urea and air bubbles.

11.

Place the upper and lower buffer tank lids onto the tanks.  Then insert the power cable on the upper buffer tank and connect it to the high voltage connector on the instrument chassis.

12.

Then start the 30 minute pre-run.  Runs are started by selecting New Run from the e-Seq File menu.  At the start of each run, e-Seq initializes the DNA sequencer, focuses the laser/microscope and starts the electrophoresis.  At the end of the pre-run, e-Seq pauses operation automatically, maintains gel temperature and waits for the samples to be loaded.

13.

To load your samples:

a.

Open the instrument door and remove the upper buffer tank lid.  

b.

Flush the well completely with buffer using the 20cc syringe to remove urea or other particulate matter that may have settled into the wells during the pre-run.

c.

Place the yellow loading paper behind the wells to help you visualize you sample being loaded into the appropriate well.

d.

Each sample should be loaded in the following base order (ACGT).  Load 0.8 ul of sample into each well using flat 0.2 mm micropipette tips and a 0.5-10 ul micro-pipettor.  Carefully position the tip between the glass plates and slowly release the mixture into the wells.

14.  Remove the visualization aid and start the electrophoresis.  Replace the upper buffer tank lid, close the door and click the Start Run button in the Scanner Console Window.  This window will now display “Run in Progress”.

Lab 1.6:  Analysis of Sequencing:  Do I have a Factor V or Factor V leiden gene?

In today’s lab, you will determine the sequence for each of your DNA samples and then compare them to determine if any of the samples contain the mutation that is characteristic of the Factor V leiden gene.

Analyzing your DNA sequencing

1.

View and Edit your gel image as required.  You may need to modify the lane order and alignment using the Modify sample Window.

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