Build-a-Gene Session 4

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LIGATION

The restriction enzyme digest that we completed last week generates DNA molecules with single stranded regions, termed "sticky" ends. When these molecules are combined, complementary bases can hydrogen bond, bringing the DNA molecules together. The enzyme DNA ligase will then create a phosphodiester bond, joining the backbone of one DNA molecule to the backbone of the other DNA molecule in a covalent bond. This step will allow us to join the vector, the promoter, and the emGFP gene together into one circular DNA molecule. For more information on restriction enzyme digests and DNA ligation: [1]


1. Combine all of the reagents below together into one thin-walled PCR tube.


Vector 1,5 ul
emGFP gene 1.5 ul
Promoter 1.5 ul
T4 Ligase + buffer mix 5.5 ul
Total 10 ul


2. Incubate at 16C for 30 min

3. Incubate at 80C for 20 min


TRANSFORMATION

This step will allow us to transfer the vector containing the emGFP gene and promoter into bacterial cells. This will separate the individual DNA molecules because almost all cells will pick up only one DNA molecule (we are therefore "cloning" the DNA). As the bacteria grow, they will continue to replicate the introduced DNA as they replicate their own bacterial chromosome. This will lead to amplification of the DNA as the cells grow. For more information on the mechanism of transformation: [2]For more information on thie protocol for transformation: [3]


1. Add 2 ul of ligation product to a prechilled tube. Into a second tube, add 2 ul of control DNA. Into a third tube, add 2 ul of water.

2. To each tube, add 100 ul of chilled bacterial culture in TSS*. Flick the tube gently.

3. Incubate on ice 30 min

4. Add 900 ul SOC media

5. Incubate at 37C for 1 hour

6. Spin down tubes and remove all of the liquid, leaving the bacterial pellet intact.

7. Resuspend the bacteria in 100 ul water.

8. Transfer to LB+Carb plates and spread the bacteria on the plates with glass beads.

9. Incubate overnight at 37C

  • Note: TSS consists of LB broth with 10% (wt/vol) PEG (molecular weight 3350 or 8000), 5% (vol/vol) DMSO, and 20-50 mM Mg2+ (MgSO4 or MgCl2), at a final pH of 6.5