PCR experiment practice for undergraduate student
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~^^ If you wonder when you first do PCR, how it looks in real experiment. Enjoy!
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PCR experiment practice for undergraduate student
- 1. Class of Modern Life Science Laboratory - May, 2014 - T.A Hong Nhung Nguyen & 서보경 Experiment 10 Polymerase Chain Reaction (PCR)
- 2. Contents ①Introduction ②Materials and Methods ③Result and Discussion ④Trouble shooting Purpose of today’s experiment What is PCR?
- 3. Purpose of today’s experiment① Introduction www.expeditions.udel.edu Our previous experiments (A) (B) (C) Purpose: Amplify a “DNA fragment of interest” in genomic DNA, total cDNA, plasmid.
- 4. Dr. Kary Mullis, the inventor of PCR, was awarded the 1993 Nobel Prize in Chemistry. PCR invention and definition① Introduction PCR is a technique which is used to amplify a specific region of DNA, in order to produce enough DNA to be adequately tested. A special DNA polymerase (Taq) is used to make many copies of a short DNA fragment (100-10,000 bp) defined by primers.
- 5. 81,267,844 bp 81,572,676 bp start end ATG10 is located at 5q14.1, size: 304,833 bases Autophagy Related Gene 10 (ATG10) in Chromosome 5 ATG10 …. PCR to amplify a DNA fragment of interest (861 bp) ① Introduction
- 6. 81,267,844 bp 81,572,676 bp start end ATG10 is located at 5q14.1, size: 304,833 bases ATG10 …. PCR to amplify a DNA fragment of interest (861 bp) Forward and Reverse primers location in genomic DNA
- 7. for PCR (to amplify a “DNA fragment of interest)Materials and Methods 1. DNA template 2. Primer (10 pmol) 3. Taq DNA Polymerase 4. dNTPs mixture (10 mM) 5. Taq Buffer 10X and 25 mM MgCl2 mixed 6. 5X band doctor (Purchased from Solgent) use only when template DNA is genomic DNA PCR machine 1. DNA (genomic DNA isolated from human or Arabidopsis) Stock concentration: 500 ng/ul 2. cDNA (complementary DNA) synthesize from RNA after Reverse Transcriptase (RT) 3. Plasmid DNA Stock concentration: 50 ug/ul Mixture of Forward and Reverse primer (Listed as detail in your protocol) Stock 5U/ul, speed of synthesize: 1 minute/1 kb
- 8. Mixture Add DNA template to each sample Experimental design and prepare the mixture PCR machine programming PCR machine Thermocycles
- 9. Methods Programming Polymerase Chain Reaction Gene Sequence (5’-->3’ direction) Template DNA Tm Expected size(kb) gDNA ATG10-F GAA CAT CCA ATA CTT GGG CAA C MCF-7 gDNA 0.86 ATG10-R AGG GAC ATT TCG TTC ATC CTG 53 53 ? Extension time
- 10. PCR in test tubes Melting 94 oC Temperature 100 0 50 Time 5’3’ 3’5’
- 12. PCRMelting 94 oC Annealing Primers 50 oC Extension 72 oC Temperature 100 0 50 Time 3’5’ 5’3’ 5’ 5’ Melting 94 oC
- 13. PCR Melting 94 oC Melting 94 oC Annealing Primers 50 oC Extension 72 oC Temperature 100 0 50 Time 30x 3’5’ 5’3’ Heat Heat 5’ 5’ 5’
- 14. PCR Melting 94 oC Melting 94 oC Annealing Primers 50 oC Extension 72 oC Temperature 100 0 50 Time 30x 3’5’ 5’3’ 5’ 5’ 5’ 5’ 5’ 5’
- 15. PCR Melting 94 oC Melting 94 oC Annealing Primers 50 oC Extension 72 oC Temperature 100 0 50 Time 30x 3’5’ 5’3’ 5’ 5’ 5’ 5’ 5’ 5’ Heat Heat
- 16. PCR Melting 94 oC Melting 94 oC Annealing Primers 50 oC Extension 72 oC Temperature 100 0 50 Time 30x 3’5’ 5’3’ 5’ 5’ 5’ 5’ 5’ 5’ 5’ 5’ 5’ 5’
- 17. Fragments of defined length PCR Melting 94 oC Melting 94 oC Annealing Primers 50 oC Extension 72 oC Temperature 100 0 50 Time 30x 3’5’ 5’3’ 5’ 5’ 5’ 5’ 5’ 5’ 5’ 5’ 5’ 5’
- 18. Why PCR product using cDNA as template sometimes smaller in size? Result PCR using ATG10 F/R specific primer with different templates Where primer should be pick to check mRNA level? 861 bp 207 bp
- 19. Gene Sequence (5’-->3’ direction) Template DNA Annealing Tm Expected size(kb) gDNA ATG10-F GAA CAT CCA ATA CTT GGG CAA C MCF-7 gDNA 0.86 ATG10-R AGG GAC ATT TCG TTC ATC CTG 53 53 http://genome.ucsc.edu Isoform 1 Isoform 2 Isoform 3 861 bp 207 bp
- 20. Expected size Common troubles in PCRTroubleshooting Contamination?? Expected size No band?? ??
- 21. Summary 1. PCR is a technique which is used to amplify a specific region of DNA, in order to produce enough DNA to be adequately tested. 2. PCR is very sensitive 3. PCR need to be well-design and perform in optimal condition Today: Amplify a “DNA fragment of interest” in genomic DNA, total cDNA, plasmid. 861 bp 207 bp