Gel electophoresis and Cloning

From OpenWetWare
Jump to: navigation, search

Gel Electrophoresis

Ligation

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.


Ligation Procedure

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]

Bacterial Transformation

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)

Transformation Procedure

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.