PCR CONCERPTS & PCR SET UP LECTURE.pptx.

importance of PCR and its uses

1. Polymerase Chain Reaction (PCR) CONCERPTS & PCR SET UP

2. What is PCR? It is a type of a reaction in which a specific gene on the DNA is amplified in a PCR machine. Or It is a type of a reaction in which copies of specific segment of a gene on the DNA are produced using a PCR machine. 2

3. REQUIREMENTS FOR A PCR REACTION PCR instruments 2. PCR Reagents 3. PCR Reaction Volume 4. Ice cubes – for placing on master mix reagents during the master mix preparation except for the Taq polymerase which has to be at -20 o C until use. 3

4. PCR INSTRUMENTS PCR machine Vortex machine Centrifuge machine 1.5ml tube PCR reaction tubes Reaction tube rack Micropipettes of different sizes Micropipette tips of different sizes 4

5. Reagents PCR master mix DNA template 5

6. PCR master mix PCR master mix is prepared in one reaction tube of 1.5ml and will contain the following . Distilled water Buffer Forward Primers Reverse Primers dNTPs Taq Polymerase – to be kept at - 20 o C until use. 6

7. Before the PCR Master mix is prepared, it is necessary to calculate the final amount (Volume) of each reagent required for a number of samples under examination. Take into account the addition of a positive control and a negative control when making the calculations. 7

8. What are primers? Short single strands of DNA having nucleotides ranging from 20 to 30. Primers anneal to the DNA template in a PCR reaction. 8

9. DNA Template e ither Genomic DNA in a Genomic PCR or Metagenomic DNA in a metagenomic PCR 9

10. Reaction Volume The researcher also need to decide on the reaction volume of the PCR to be prepared in each PCR tube. In a thermo cycler PCR the reaction volume range from 10µl to 200 µl 10

11. PCR SET UP The first step is to prepare the PCR master mix PCR master mix is prepared from a PCR room (clean room) Put on a clean lab coat and remove the one you were wearing during DNA extraction. Put on clean disposable gloves No samples of DNA should ever enter the PCR master mix preparation room. 11

12. Rooms DNA extraction room Master mix preparation room PCR work station room PCR Machine and gel room 12

13. Clean the work surface with jik and finally with alcohol. Remove all the required reagents from the fridge except the Taq polymerase as it has to be kept at -20 C. 13

14. Keep all reagents on ice for the duration of the master mix preparation. To ensure homogenous distribution of PCR reaction, it is advisable to prepare the mix in a single reaction tube (in one tube) labelled master mix. 14

15. Vortex and spin reagents Vortex reagents in a vortex machine and spin each reagent in a centrifuge machine before use to avoid pellet of material forming in the tube. 15

16. Labelling PCR tubes Prepare and label the PCR tubes according to the number of samples to be tested Include the positive and negative control Label tubes on the lid and sides Place the labelled tubes on the reaction tube rack. 16

17. Micropipettes Using the different micropipette sizes and tips, measure the calculated volume of each reactant and place in a reaction tube labelled master mix. 17

18. Add the required PCR reagents to the master mix tube as they are listed in the protocol using a different pipette and a different tip. Finally add the T aq enzyme to the master mix tube. When all the master mix reagents are included in the master mix tube, Vortex and spin briefly to mix the preparation. 18

19. Aliquoting the master mix Take the prepared master mix to the PCR work station. Also take the DNA samples to the PCR work station First, aliquot the necessary amounts of the master mix into each single PCR tube as per calculation. This should be done in a PCR work station. 19

20. For example, in a PCR reaction requiring 12µl of the master mix, place 12µl of the master mix in each labelled PCR tube using the same pipette and same pipette tip. Check that you have added the same amount of the master mix to all the PCR tubes by lifting the tubes up . Put the tubes on the rack to add the required amounts of DNA to each tube as per calculation. 20

21. Open one tube at a time to add DNA and close after adding DNA Add DNA to the tubes using the same micropipette but using a different tip for each sample to avoid mixing the samples. Then to each tube, add the required amount of the DNA template in this case it will be 8µl in a 20µl PCR reaction in each tube. 21

22. PCR running Take the preparation into the PCR machine. Set the PCR machine according to the desired temperatures and time for DNA denaturing, Primers annealing and elongation time and the number of cycles. View the PCR products on agarose gel. 22