Gel electophoresis and PCR purification

Pouring Gels

We need to check how well our assembly worked by running our PCR products (and controls) on an agarose gel to verify whether we have assembled a significant amount of the full-length gene. Remember, your positive controls should each produce a DNA product and your negative controls should not produce a DNA product.

Pouring a Gel:

1. Weigh out 0.5 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.
3. Allow the agarose 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 1 ul of 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 the gel to solidify for 15-20 minutes. During this time, you prepare your samples (below).

Gel electrophoresis

Preparing your samples:

1.Gather your finish PCR products from last session. Transfer each to a 1.7 ml microcentrifuge tube and label each tube with your initials and either “F-PCR PC”, “F-PCR NC”, or “GFP gene”. Obtain your templateless PCR controls that have been stored in the fridge.
2.Obtain 5 microcentrifuge tubes and label with numbers 1-5.
3.To each tube, add 5 ul of water.
4.To each tube, add 2 ul of 6X DNA loading dye.
5.To each tube, add 5 ul of the appropriate PCR product:

• T-PCR PC
• T-PCR NC
• F-PCR PC
• F-PCR NC
• GFP gene

Running a Gel:

1. Into lanes 1-5, load 11 ul of each of your PCR products (mixed with water and dye).
2. Into lane 6, load 5 ul of the DNA ladder.
3. Place the lid with electrodes onto the gel box, and set voltage to 100V.
4. Run gel approximately 30 minutes or until the dye is 2/3 of the way down the gel, then take a picture.
5. When done, you want to check that:

• There are no DNA bands in either of the NC lanes of the gel
• There are DNA bands in the PC lanes of the gel. These should be ~500 base pairs in length. Size can be estimated by comparing the migration of the DNA band with the DNA bands of reference ladder; the sizes of the DNA products on the reference ladder are pictured at right.
• The GFP gene has assembled and amplified. There should be a DNA band of ~750 base pairs in that lane of the gel.

 PCR purification

The next step is to combine the GFP gene, the promoter which is necessary for the gene to be expressed in cells, and a vector which will allow the gene to be maintained in cells. Before we can join the DNAs together by Gibson Assembly, we must remove the old buffers and proteins using a purification kit. We will purify the GFP gene that you just amplified and you will be given the purified promoter and vector.

1. Combine your PCR products with 80 ul of water and mix.
   
2. Add 500 ul buffer PB and pipet up and down several times to mix.
3. Obtain a spin column/collection tube and label with your initials. Add the liquid from step 2 to the top of the column.
4. Spin in centrifuge for 1 min (be sure the centrifuge is balanced with another tube on the opposite side).
5. Remove the spin column and pour out the liquid from the bottom collection tube. Put the spin column back in the collection tube.
6. Add 750 ul buffer PE to the top of the column.
7. Spin in centrifuge for 1 min (be sure the centrifuge is balanced with another tube on the opposite side).
8. Remove the spin column and pour out the liquid from the bottom collection tube
9. Repeat steps 6 and 7.
10. Obtain a 1.7 ml microcentifuge tube and label with your initials and “GFP gene”.
11. Discard the collection tube and transfer the column to the new labeled microcentrifuge tube.
12. Add 50 ul TE to the column. The microcentrifuge tube cap will not close over the spin column, just leave the tube open.
13. Wait 1 min and then spin the column in centrifuge for 1 min.
14. Throw out column. The liquid in the bottom of the microcentrifuge tube is your purified DNA.