PCR Teaching kit         

Cat# BB-MTK010S (5Reactions) 
Cat# BB-MTK010 (10Reactions) 
 

Objectives:

• To perform PCR amplification of specific target sequence from template
• To analyze the amplified product by Agarose gel

Principle: PCR (Polymerase Chain Reaction) is an in vitro method of enzymatic synthesis of specific DNA sequence, developed by Kary Mullis in 1983. It is a very simple technique for characterizing, analyzing & synthesizing any specific DNA or RNA from any source.

PCR consists of the following three basic steps:

Denaturation: During this step, two strands melt (open) to form single stranded DNA. This step is generally carried out at 92^oC-96^oC.

Annealing: In this step, primers anneal to each original strand of the template DNA for new strand synthesis. This step is carried out at 45^oC to 60^oC.

Extension: At this step the DNA polymerase    synthesizes a new DNA strand complementary to the template DNA strand by adding dNTPs that are complementary to the template DNA in 5′ to 3′ direction, condensing the 5′-phosphate group of the dNTPs with the 3′-hydroxyl group at the end of the nascent (extending) DNA strand.

Materials provided: (for 10 reactions)

Procedure:

Add the reagents to the PCR tubes in the following order:

Mix the reagents properly, and place the tube in the PCR  Machine

PCR Amplification: Carry out the amplification in a thermo cycler for 30 cycles using the following conditions:
Preparation of 1% Agarose gel: 

1. Prepare the gel casting tray by sealing the two ends with adhesive tape.
2. Put the comb in the appropriate place of the gel casting tray.
3. Prepare 1 X TAE by diluting appropriate amount of 50 X TAE Buffer (for carrying out one experiment approximately 200 ml buffer is needed).
4. Weigh 0.5gm of Agarose and add 50ml of 1X TAE for preparing 1% Agarose
5. Boil until the agarose dissolves completely, cool it to around 50⁰C, add 5µl EtBr to the agarose solution and mix
6. Pour the Agarose solution slowly to avoid generation of any air bubble .Keep the gel  undisturbed at room temperature till the agarose solidifies.
7. Pour 1X TAE into the gel tank till the buffer level stands at 0.5 to 0.8 cm above the gel
8. Gently lift the comb, ensuring the wells remain

Analysis on Agarose gel and Observation:

• Add 5ul of 6X DNA loading Dye in to the PCR tube and mix
• Carefully pipette 5µl of reaction mixture and load into the well of 1% Agarose
• Load 5µl of 1Kbp DNA ladder (ready to use) Run the sample at 100 volts till the blue dye front reaches 3/4^th length of the gel.

Adapted from internet