Biotech Lab Protocols, Tips & Tricks

PURPOSE: To perform ligation of given plasmid DNA(vector,insert) to use it for transformation.

REAGENTS: Plasmid DNA fragments , T4 DNA ligase , Ligase buffer , Wash buffer and Elution buffer.

INSTRUMENTS: Microcentrifuge, Heating block.

PRINCIPLE: DNA ligation is the process of joining together two DNA molecule ends (either from the same or different molecules). This process is accomplished both invitro and invivo by the enzyme DNA Ligase. It involves

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creating a phosphodiester bond bond between the 3' hydroxyl of one nucleotide and the 5' phosphate of another. It can repair single stranded nicks in a double stranded DNA and join the double stranded DNA resctriction fragments having either “blunt” ends or “homologous cohesive” ends. Typically, it is easier to ligate molecules with complementary sticky ends than blunt ends.

T4 DNA ligase: It is approximately 60,000dalton protein produced by bacteriophage T4 requiring ATP as energy source. T4 DNA ligase is the most commonly used DNA ligase for molecular biology techniques and can ligate 'sticky' or blunt ends.

REACTION MIX:

20μL Ligation Mix(1:1 ratio)

1) 5 μL vector

2) 5μL insert

3) 2μL Ligase Buffer

4) 7.5μL deionized H2O

5) 0.5Ligase Enzyme

PROCEDURE: For ligation we will use 1:1 and 11:3 ratio. Ligation will be performed at 4 degree C (optimum) and 25- 28 degree C. The amount of respective Dna will be calculated using the following formula:

1. The appropriate amount of deionized H2O was added to sterile tube.

2. The ligation buffer was added to the tube.

NOTE: Vortex buffer before pipetting to ensure that it is well-mixed. Remember that the buffer contains ATP so repeated freeze, thaw cycles can degrade the ATP thereby decreasing the efficiency of ligation.

3. The appropriate amount of insert was added to the tube.

4. The appropriate amount of vector was added to the tube.

5. Ligase enzyme was added.

NOTE: Vortex ligase before pipetting to ensure that it is well-mixed. just touch your tip to the surface of the liquid when pipetting.

6. The ligation mix was kept at 4°C for overnight or at room temperature for 2-3hrs

RESULT: The ligation mix was stored at 4°C overnight for ligation to take place.

INFERENCE: Change in concentration of insert and vector may effect the ligation process.If reaction is set up at higher temperatures annaeling of the ends becomes difficult, while lower temperatures diminishes the ligase activity.