Dave Gray's Build-A-Gene Class Notes - Session 3
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.
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.
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:
- NEB4 - NEBuffer 4 from New England Biolabs.
- BSA - Bovine serum albumin, a protein extracted from the blood of cows. Used to stabilize some enzymes during digestion of DNA and to prevent adhesion of the enzyme to reaction tubes, pipet tips, and other vessels.
- DpnI - A restriction enzyme which digests methylated DNA
Then we selectively add the following to one or more of our vials:
- XbaI - A restriction endonuclease from Xanthomonas badrii that recognizes the sequence T^CTAG_A.
- PstI - A Type II restriction endonuclease (or restriction enzyme) from Providencia stuartii that recognizes the sequence C_TGCA^G.
- EcoRI - A restriction endonuclease from E. coli RY13 that recognizes the sequence G^AATT_C.
- SpeI - A restriction endonuclease from Sphaerotilus species that recognizes the sequence A^CTAG_T.
Finally, the vials were placed into an incubator, floating in a warm bath in small foam plastic floats.