Dave Gray's Build-A-Gene Class Notes - Session 3

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Next, we need to prepare our three segments of DNA for joining.  This requires "cutting" them at specified points, leaving "sticky" ends.  To do this, we combined 10 ul of each of our products in new tubes and added to each a mix of water plus three other items:
Next, we need to prepare our three segments of DNA for joining.  This requires "cutting" them at specified points, leaving "sticky" ends.  To do this, we combined 10 ul of each of our products in new tubes and added to each a mix of water plus three other items:
 +
# NEB4
 +
# BSA
 +
# Dpnl
 +
 +
Then we selectively add the following to one or more of our vials:
 +
 +
# Xbal
 +
# Pstl - A Type II restriction endonuclease (or restriction enzyme) from Providencia stuartii.
 +
# EcoRI
 +
# Spel

Revision as of 20:37, 15 August 2013

In session 3, we accomplished several things:

1. We made our own gel for electrophoresis. 2. We used gel electrophoresis to check the results of our PCA work to build and amplify a complete emGFP gene in Session 2. 3. We purified the DNA. 4. We added restriction enzymes to cut the promoter, vector and emGFP coding sequence in preparation for jointing them together.

Preparing the Gel

To make the gel, we mixed .5 g of agarose (a polymer extracted from seaweed) with TAE buffer (a solution containing a mixture of Tris base, acetic acid and EDTA) and heated this to dissolve the agarose. After cooling, we added Gel Red (how much?) and poured this into plastic trays to solidify. The trays have a plastic "comb" that forms "wells" where we add the DNA. Once the gel solidified, we removed the "comb", placed the tray into the gel box and covered with TAE buffer.


Gel Electrophoresis

We mixed a bit of loading dye and water with 5 ul of the emGFP genes we built and with our control. In the first lane of the gel, we placed 10 ml of the DNA ladder. The product and control were placed in the next two lanes. Then we turned on the electricity.


Purification

This process removes the buffers and proteins left over with our DNA vector, promoter and emGFP coding gene. We used the Qiaquick PCR purification kit to do this for each of these separately. It uses a silicon filter and a few salt solutions. Initially, the "PB" solution is added which causes the DNA to stick to the silicon filter but allows the other material to pass through. We spin in the centrifuge, add the PE buffer and spin again - then repeat with PE. With each of these cycles, we throw away the liquid that passed through the filter. Next we add TE solution and spin once more. This releases the purified DNA and we keep the results.


Cutting with Restriction Enzymes Next, we need to prepare our three segments of DNA for joining. This requires "cutting" them at specified points, leaving "sticky" ends. To do this, we combined 10 ul of each of our products in new tubes and added to each a mix of water plus three other items:

  1. NEB4
  2. BSA
  3. Dpnl

Then we selectively add the following to one or more of our vials:

  1. Xbal
  2. Pstl - A Type II restriction endonuclease (or restriction enzyme) from Providencia stuartii.
  3. EcoRI
  4. Spel



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