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Preparation and steps of PCR experiment

Views: 12     Author: Site Editor     Publish Time: 2021-11-18      Origin: Site

Step composition:

① Denaturation of template DNA: After the template DNA is heated to about 94℃ for a certain period of time, the template DNA double-stranded or the double-stranded DNA formed by PCR amplification is dissociated to make it single-stranded so that it can be combined with the primer. Prepare for round reaction;

② Annealing of template DNA and primer (renaturation): After the template DNA is denatured into single-stranded by heating, the temperature drops to about 55°C, and the primer is paired and combined with the complementary sequence of the single-stranded template DNA;

③ Primer extension: DNA template---primer conjugate under the action of enzyme, using P as the reaction material and target sequence as the template, according to the principle of base pairing and semi-reserved replication, a new half complementary to the template DNA strand is synthesized Keep the copy chain.

Repeat the cycle of denaturation-annealing-extension three processes, you can get more "semi-reserved replication chain", and this new chain can become the template for the next cycle. It takes 2 to 4 minutes to complete a cycle, and the target gene to be amplified can be amplified several million times in 2 to 3 hours.

Experimental reagents and equipment:

Template DNA, 2.5mmol/L q DNA polymerase (5U/SSR primer

10 ×buffer, 15mmol/L Mg2+, ddH2O

PCR machine, pipette, PCR plate

thermal cycler

Experimental steps:

1. Experimental equipment and materials:

1, pipette: 1ml, 200μl, 20μl, 10μl, 2μl

2, Tips: 1ml, 200μl, 20μl

3, homogenization tube: 5ml

4. Tip table: one with 1ml tips and one with 20μl tips

5, EP tube: 1.5ml, 0.2ml, 100μl

6. Reagent bottles: two 60ml brown reagent bottles (wide mouth, with lid)

1 125ml white reagent bottle (put absolute ethanol)

7, measuring cylinder: 50ml, 250ml, 500ml

8. Volumetric flasks: 250ml, 500ml, 1000ml

9. Test tube rack: 5ml, 1.5ml, 20μl

10. Saline bottle: 250ml and 500ml each for 2 spares, one for absolute ethanol and the other for DEPC water

11, aluminum lunch box: 4

12. Small plastic lunch box: 1

13, large porcelain jar: 2

14, tin park paper: one roll

15, roll paper: 2 rolls

16. Erlenmeyer flask: with lid, slightly larger

Handling and preparation of experimental equipment:

1. Plastic products: (including pipe tips, EP tubes, homogenization tubes, etc.)

Pour the DEPC water from the volumetric flask into the porcelain jar, and soak the plastic products one by one. Among them, the small pipette needs to be injected into the DEPC water, overnight, then high pressure, and then bake it for use. Put the pipette tip into the suction pipe before the experiment. Head stage, high pressure again (EP tube)

2. Glass products: soak in acid overnight, rinse well, and dry the tin foil for later use (DEPC blister) (after washing, soak 1‰ DEPC overnight, then bake it)

3. Homogenizer: (including scissors and tweezers) after washing first, then high pressure (no need to soak DEPC)

Reagent preparation:

1. DEPC water: aspirate 1ml and place in 1000ml double distilled water to make 1‰ DEPC water, put it in a 1000ml volumetric flask and let it stand for 4 hours.

2. 75% ethanol: mix with absolute ethanol and DEPC water, and then store at -20°C (the DEPC water needs to be high-pressure first)

3. Isopropanol: put it in a brown bottle

4: Put it in the brown bottle

5. Agarose

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