Gel electophoresis, Ligation, and Transformation
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.
Preparing your samples:
1. Gather your finish PCR products from last session. Transfer each to a microcentrifuge tube and label each tube with your initials and either “Gene 68”, “F-PCR PC”, or “F-PCR NC.
2. Obtain your templateless PCR controls that have been stored in the fridge.
3. Obtain 5 microcentrifuge tubes and label with numbers 1-5.
4. To each tube, add 2 ul of 6X DNA loading dye.
5. To each tube, add 10 ul of the appropriate PCR product:
1.Gene 68 2.F-PCR PC 3.F-PCR NC 4.T-PCR PC 5.T-PCR NC
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 10 ul of the DNA ladder (this is premixed with water and dye).
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 (lanes 3 and 5).
• There are DNA bands in the PC lanes of the gel (lanes 2 and 4). These should be ~750 base pairs in length. Size can be estimated by comparing the migration of the DNA band with the bands of the DNA ladder (which is included below).
• Gene 68 has assembled and amplified. There should be a DNA band of 426 base pairs in that lane of the gel (lane 1).
The PCR reaction produces millions of DNA molecules-some have the correct 426 base pair sequence, but others are not perfect (remember that each step in chemical synthesis is only 99% accurate). We only want to continue with the perfect ones, so we need to separate the DNA molecules to identify those that are correct. To do this, we will perform DNA cloning, where we ligate the PCR products into a plasmid vector (a segment of DNA that will hold the PCR product) and then transform the ligated DNA into bacterial cells. Importantly, each bacterial cell will pick up only one piece of ligated DNA, separating the individual DNA molecules from each other so that we can determine which are correct and which are not.
1. Obtain your assembled Gene 68 PCR product. Transfer 1 ul to a new microcentrifuge tube.
2. To this tube, add the following components in the order listed:
1 ul of pMiniT vector
3 ul of water
4 ul of Cloning mix 1
1 ul of Cloning mix 2
3. Incubate the tubes at room temperature for 15 min. [Continue to step 1 below while waiting]
For the transformation of bacteria, you will be performing three reactions:
• Transformation of the DNA that you ligated above
• A positive control called pUC19 (this DNA should transform bacteria at high efficiency)
• A negative control (water; should not transform bacteria unless contaminating DNA is present)
1. Obtain 3 microcentrifuge tubes and label with your initials and numbers 1-3. Prechill the tubes by placing them on ice.
2. To the first tube, add 1 ul of the ligated DNA
3. To the second tube, add 1 ul of pUC19 DNA (positive control)
4. To the third tube, add 1 ul of water
5. To each tube, add 25 ul of chilled bacterial cells. Flick the tube gently.
6. Incubate on ice for 20 min
7. Heat shock the cells by placing at 42C for 30 seconds (timing is critical here!!)
8. Immediately place the tubes back on ice for 5 min
9. Add 250 ul of sterile SOC media. Incubate the cells at 37C for 60 minutes.
10. Pipet the bacteria onto the center of an LB+Amp plate. Add 6-7 sterile glass beads and use these to spread the bacteria on the plates. Incubate the plates overnight at 37C.