Build-a-Gene Session 3

This week we will be checking to see whether our emGFP gene was successfully assembled and amplified and then we will be purifying our promoter, vector and coding sequence. Once they are purified, we will cut the promoter, vector and coding sequence with restriction enzymes so that the three pieces can be joined together and transferred into E. coli cells in our next session.

GEL ELECTROPHORESIS

Now, we need to check how well our PCR reaction worked by running our PCR product on an agarose gel to verify whether we have amplified a significant amount of the promoter sequence.

Pouring a Gel:

1. Weigh out 0.35 g of agarose on a piece of weigh paper. Transfer to an Erlenmeyer flask. Add 50 ml of 1x TAE. 2. Place the flask in the microwave and heat until the agarose is completely transparent and colorless. You may need to remove the flask from the microwave and swirl the mixture a few times. 3. Remove the flask of clear agarose and allow it to cool. This will take about 10 min. 4. While the agarose is cooling, place the gel tray into the gel box and add the comb. 5. When the agarose is cool, add Gel Red to the melted agarose. 6. Swirl the agarose to incorporate the Gel Red and pour the agarose into the gel tray. 7. Allow at least 20 minutes for the gel to solidify. Once solid, carefully remove the comb and place the solidified gel into the gel box so that the wells are oriented on the same side as the black electrode. 8. Add enough 1x TAE buffer to completely cover the gel by about 1 cm.

Preparing your samples:

1. Transfer each of your PCR products to a new tube and label each tube with the sample name and your initials. Be sure to include your control. 2. On a piece of parafilm, spot out 2 ul of 5x DNA loading dye with a P20 pipet. 3. Add 3 ul of water to the spot of dye. 4. Add 5 ul of your PCR product to the spot.

Running a Gel:

1. Into the first lane of the gel load 10 ul of the DNA ladder (pre-mixed with water and dye). 2. Into lanes 2, load 10 ul of your PCR product (mixed with water and dye). 3. Place gel lid with electrodes on gel box, and turn on the voltage. 4. Run gel approximately 30 minutes or until the dye is 2/3 of the way down the gel, then take picture.

PCR PURIFICATION

The next step is to cut the emGFP gene, the promoter, and the vector with restriction enzymes so that they can be joined together. Before we can cut the DNAs we must remove the old buffers and proteins using the Qiaquick PCR purification kit (Information on the Qiaquick purification technique and protocol is here: [1], focus on p. 11-13 and 19-20). We will purify the emGFP gene that you just amplified as well as the promoter and vector PCR reactions.

1. Combine your PCR products with 60 ul water and mix.
2. Add 400 ul buffer PB and mix.
3. Add the liquid to the top of a column
4. Spin in centrifuge for 1 min and pour out liquid from the bottom
5. Add 750 ul buffer PE
6. Spin in centrifuge for 1 min and pour out liquid from the bottom
7. Repeat step 6 and transfer column to new tube.
8. Add 50 ul TE to the column. Wait 1 min. Spin in centrifuge for 1 min. Throw out column and keep the liquid in the bottom, which is your purified DNA.

RESTRICTION ENZYME DIGEST

In order to join the vector, the promoter, and the emGFP gene together, we must cut each with restriction enzymes to create overlapping ends that can be joined together. Information on how restriction enzymes work can be found here: [2]

1. Transfer 10 ul of each of your purified DNAs(your vector, promoter and emGFP gene) to a new labeled tube.

2. Make a master mix containing all of the components that will be in every reaction. Combine each of the following into one tube:

H2O	20 ul
10X NEB4	12.5 ul
10X BSA	10 ul
DpnI	2.5 ul

2. Now add 9 ul of your master mix to each of your three tubes containing DNA (your vector, promoter and emGFP gene).

3. Add enzymes according to the table below to each of your tubes:

DNA	vector	emGFP gene	promoter
XbaI	0	0.5 ul	0
PstI	0.5 ul	0.5 ul	0
EcoRI	0.5 ul	0 ul	0.5 ul
SpeI	0 ul	0 ul	0.5 ul

Information on why we cut with these enzymes is available on page 1 here: [3](Our promoter is the "upstream part", our emGFP gene is the "downstream part" and our vector is the "destination plasmid").

4. Incubate at 37C for 2 hours and then incubate at 80C for 20 min.