Table of contents :
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| size range (kb) | pulse time (s) | electrophoresis time (h) |
| 5-50 | 1 | 10 |
| 20-100 | 3 | 10 |
| 50-250 | 10 | 14 |
| 100-400 | 20 | 14 |
| 200-1,000 | 45 | 18 |
| 200-1,600 | 70 | 20 |
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| first cycle | 4 min at 94°C | 1 min s at 42-46°C | 1 min at 72°C |
| 24 cycles | 40 s at 94°C | 1 min s at 42-46°C | 1 min at 72°C |
| last cycle | 40 s at 94°C | 1 min s at 42-46°C | 5 min at 72°C |
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| 24 cycles | 40 s at 94°C | 90 s at 72°C |
| last cycle | 40 s at 94°C | 5 min at 72°C |
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| 1 | 1-2 min at 93°C | ||
| 2-31 (see step 7) | 50 s at 92°C | 1 min at 50-58°C | 10 min at 63°C |
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| plasmid | 18-22 |
| bacterial | 25-30 |
| mammalian | 35-40 |
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| 1 | 5 min at 94°C | 5 min at 50-58°C | 40 min at 72°C |
| 2-31 | 40 s at 94°C | 1 min at 50-58°C | 3 min at 72°C |
| 32 | 40 s at 94°C | 1 min at 50-58°C | 15 min at 72°C |

| fluorophores |
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| Alexa Fluor 350 |
346
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442
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| Alexa Fluor 430 |
434
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539
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| Alexa Fluor 488 |
495
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519
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| Alexa Fluor 532 |
531
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554
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| Alexa Fluor 546 |
556
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573
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| Alexa Fluor 555 |
555
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565
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| Alexa Fluor 568 |
578
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603
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| Alexa Fluor 594 |
590
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617
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| Alexa Fluor 610-R-PE |
496, 546, 565
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630
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| Alexa Fluor 647 |
650
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668
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| Alexa Fluor 647-R-PE |
496, 546, 565
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668
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| Alexa Fluor 660 |
663
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690
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| Alexa Fluor 680 |
679
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702
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| Alexa Fluor 680-R-PE |
496, 546, 565
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702
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| Alexa Fluor 700 |
696
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719
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| Alexa Fluor 700-R-PE |
650
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723
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| Alexa Fluor 750 |
752
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779
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| Alexa Fluor 750-R-PE |
650
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775
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| BODIPY FL | ||||
| BODIPY 564/570 | ||||
| BODIPY 558/568 | ||||
| BODIPY 594 | ||||
| BODIPY TMR-X | ||||
| Cascade Blue |
400
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420
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| CY3 | ||||
| CY5 |
649 (633 commonly used)
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670
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| CY7 |
743
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767
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| fluorescein |
494
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518
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| fluorescein isothiocyanate (FITC) |
495 (488 commonly used)
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525
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| Marina Blue |
365
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460
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| Oregon Green 488 |
496
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524
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| Oregon Green 514 | ||||
| Pacific Blue |
410
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455
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| Peridinin chlorophyll protein (PerCP) |
488
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677
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| phycobiliproteins / biliproteins are homologous chromoproteins that have covalently bound phycobilin chromophores and constitute the phycobilisomes, the light harvesting complexes of the photosynthetic apparatus in Cyanobacteria (C), Rhodophyta (R) and Cryptophyta. The phycobilisomes constitute up to 50% of the total protein in Cyanobacteria and their content in PC or PE can be up- or down-regulated by using different light conditions (chromatic adaptation). | phycoerythrin (PE) carries covalently bound phycoerythrobilin (PEB) and phycourobilin (PUB) as chromophoric groups and is composed of 2 subunits, a and b, with Mr of 14000 and 17000, respectively | C-phycoerythrin (C-PE) |
565 (488 commonly used)
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576
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| R-phycoerythrin (R-PE) |
496, 546, 565
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578
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| phycocyanin (PC) carries covalently bound phycocyanobilin (PCB) as chromophoric group and is composed of 2 subunits having a similar Mr of 21000 | C-phycocyanin (C-PC) | |||
| R-phycocyanin (R-PC) | ||||
| allophycocyanin (APC) |
650 (633 commonly used)
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660
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| phycoerythrocyanin carries covalently bound phycoviolobilin as chromophoric group |
565
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| Proteins : when a protein is tagged by fusion to a fluorescent protein, interactions between fluorescent proteins can undesirably disturb targeting or function. Unfortunately, all wild-type yellow-to-red fluorescent proteins reported so far are obligately tetrameric and often toxic or disruptive. The first true monomer was mRFP1 | green fluorescent
protein (GFP) from Aequorea
victoria (jellyfish) : 238 amino acids, with a chromophore consisting
of the phenolic side chain of Tyr66 and the imidazolidinone
ring formed by cyclization of Ser65-Tyr66-Gly67.
The chromophore is located at the center of a cylinder-shaped 3D structure
with a Ø = 24 Å and height = 42 Å.
Web resources : |
photoactivatable GFP (PA-GFP) is monomeric and gives 100-fold fluorescence contrast, can be applied for protein tracking. |
498 (488 commonly used) (blue light)
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516 (green light)
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| GFP mutants with absorption and emission bands ranging from the violet to the red spectrum of the spectrum | W399X | |||
| enhanced GFP (eGFP / EGFP) : improved brightness after blue-light excitation with respect to wild-type GFP. After irradiaton with l ~ 400 nm, the chromophore undergoes photoconversion to anionic form : as mutant T302H has a very low minor absorbance peak, it is photoinducible at following irradiations with lmajor absorption peak. |
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enhanced
cyan fluorescent protein (ECFP)
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| enhanced yellow fluorescent protein (EYFP) | ||||
| monomeric red fluorescent protein 1 (mRFP1 / DsRed1) derived from Discosoma striata (sea anemone) by directed evolution first to increase the speed of maturation, then to break each subunit interface while restoring fluorescence, which cumulatively required 33 substitutions. The latest red version matures more completely, is more tolerant of N-terminal fusions and is over tenfold more photostable than mRFP1. 3 monomers with distinguishable hues from yellow-orange to red-orange have higher quantum efficienciesref. |
277, 335, 487, 530 and 558
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583
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| mPlum was obtained by iterating SHM |
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| DsRed-E5 is a mutant of the RFP drFP583 that changes its fluorescence from green to red over time, enabling accurate monitoring of the temporal ("fluorescent timer") and spatial pattern | ||||
| photoactivatable protein Kaede | ||||
| DsRed 'greening' technique | ||||
| kindling fluorescent proteins | ||||
| photoswitchable cyan fluorescent protein (PS-CFP) is a dual-color monomeric protein capable of efficient photoconversion from cyan to green, changing both its excitation and emission spectra in response to 405-nm light irradiation. Complete photoactivation of PS-CFP results in a 1,500-fold increase in the green-to-cyan fluorescence ratio, making it the highest-contrast monomeric photoactivatable fluorescent protein described to date. At moderate excitation intensities, PS-CFP can be used as a pH-stable cyan label for protein tagging and FRET applicationsref. | ||||
| tetramethylrhodamine | ||||
| Texas Red-X |
595-596
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615-620
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| binding directly to DNA | 7-aminoactinomycin D (7-AAD) |
546 (488 commonly used)
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650
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| propidium iodide (PI) |
305 (488 commonly used)
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617
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| EMA | ||||
| Hoeschst 33342 | ||||
| binding directly to RNA | thiazole orange | |||
| tandem conjugates of 2 fluorochromes in which the energy is transferred from the first to the second, creating a larger Stoke's shift | PE-CY5 |
565 (488 commonly used)
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670
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| PE-CY7 |
565 (488 commonly used)
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767
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| PE-TxRed |
565 (488 commonly used)
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516
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| APC-CY7 |
650 (633 commonly used)
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767
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| 1 | 2 min at 94°C | 30 s at 60°C | 30 s at 72°C |
| 2-39 | 30 s at 94°C | 30 s at 60°C | 30 s at 72°C |
| 40 | 30 s at 94°C | 30 s at 60°C | 5 min at 72°C |
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| 1-30 | 15 s at 94°C | 30 s at 42°C | 15 s at 72°C |
| 31 | 15 s at 94°C | 30 s at 42°C | 2 min at 72°C |

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