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Practical molecular biology

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Presentation on theme: "Practical molecular biology"— Presentation transcript:

1 Practical molecular biology
PD Dr. Alexei Gratchev Prof. Dr. Julia Kzhyshkowska Prof. Dr. W. Kaminski

2 Course structure 10.10 Plasmids, restriction enzymes, analytics
11.10 Genomic DNA, RNA 12.10 PCR, real-time (quantitative) PCR 13.10 Protein analysis IHC 14.10 Flow cytometry (FACS)

3 PCR Thermostable DNA polymerase Oligonucleotides dNTPs Buffer Template
Cycling 3

4 PCR Detection of pathogens Detection of mutations
Person identification Cloning Mutagenesis and may more… 4

5 Quantification by PCR Ideal PCR
M=m*2N, m – starting amount of template, N-number of cycles 30 cycles =230 ≈109 40 cycles ≈1012 5

6 Quantification by PCR Real PCR
M ≈ m*2N, only in the beginning of the reaction Critical factors Size of the product Mg concentration Oligonucleotide conc. dNTPs conc. 6

7 “End point” PCR 7

8 Real-time PCR threshold Ct 8

9 Real-time PCR threshold Ct 9

10 Quantification by PCR Measure the amount of the product after every cycle Determine threshold cycle (Ct) value for each sample Calculate the amount of the product Note: Ct can be a fraction 10

11 Real-time data collection
Intercalating dyes Cheap Low specificity Can measure only one gene per tube Molecular beacons TaqMan® probes Highly specific Several genes can be measured in one tube (Multiplex PCR) Expensive Multiplex PCR is hard to optimize 11

12 Intercalating dyes SYBR Green Data collected after synthesis step 12

13 Intercalating dyes Denaturation analysis is needed for specificity analysis One peak indicates that the reaction was specific. 13

14 Fluorescence detection
FAM 14

15 Fluorescence resonance energy transfer - FRET
FAM Q 15

16 Molecular beacons Data collected during annealing step 16

17 TaqMan® probes Data can be collected anytime 17

18 Real-time PCR equipment
Light sources Laser LED Array Focused halogen lamp Halogen lamp Detectors PMT (Photo Multiplier Tube) CCD camera Light source PMT 18

19 Multiplexing 19

20 Experiment planning Selection detection method Intercalating dye
Molecular beacon TaqMan® probe Selection of house keeping gene GAPD beta actin Selection of quantification method absolute (Standard curve) relative (ddCt) 20

21 Absolute quantification
The amount of template is measured according to the standard curve – serial dilutions of known template (plasmid). Problem! Standard curve takes too much space on the plate. 21

22 Relative quantification of ID3
dCt(A)= Ct(ID3 in A) - Ct(GAPD in A) dCt(B)= Ct(ID3 in B) - Ct(GAPD in B) ddCt = dCt( A) – dCt(B) Relative Expression = 2 -ddCt Problem! ddCt method can be used only if both reaction (for ID3 and GAPD) have the same efficiency. 22

23 Relative quantification
For ddCt the slopes of standard curves for gene of interest and house keeping gene must be the same. 23

24 Relative quantification
duplicates quadruplicates 24

25 Relative quantification
Pipetting strategy 25

26 Questions?

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