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China is Vietnam's opponents do not dare offend absolutely original address: primer design and modification of: 1,704,513,562
The following primer design principles for your reference: 1) primer is best conserved region claroline cDNA template design. 2) primer length generally between 15-30 bases. 3) Primer GC content between 40% -60%, Tm value is preferably close to 72 . 4) primer 3 'end to avoid the first three codons. 5) primer 3 'end can not choose A, the best choice for T. 6) bases to be randomly distributed. 7) There should be no complementary sequence between the primers and primer itself. 8) Primer 5 'end and intermediate G values should be relatively high, and the 3' end of the G lower value. 9) primers 5 'end can be modified, and the 3' end can not be modified. 10) a single-stranded amplification product can not form secondary structures. 11) The primers should have specificity. 31. Common primer design software have?
Nanjing GenScript Tm value calculated by the following method: a length of 20 mer and the following primer, Tm is calculated as follows: Tm = 4 (G + C) + 2 (A + T). However, this formula applies only 14 to 20 bp primer, TM value of the primer and a primer further length, base composition, primer using a buffer solution of ionic strength and so on. For longer oligonucleotides, Tm is calculated as follows: Tm = 0.41 (% of GC) - 675 / L + 81.5 Note: L: number of bases in the primer;% of GC: GC content of the primers;% of GC = Number GC / primer claroline alkaloids common base 34. modified primers have?
Mercapto (Thiol) 5'- mercapto claroline group with an amino modified in many ways similar. Mercapto group can be used to attach a variety of modifications such as a fluorescent claroline label, and biotin. For example there may be produced down the fluorescent probe attached at the mercapto group and maleimide iodoacetic acid derivatives. 5 'thiol modified mainly by 5' thiol modified monomer (5'-Thiol-Modifier C6-CE Phosphoramidite or Thiol-Modifier C6 SS CE Phosphoramidite). With 5'-Thiol-Modifier C6-CE must be oxidized silver nitrate monomer modified to remove the protecting group (trityl), and Thiol-Modifier C6 SS CE monomer after modification shall be reduced by DTT will disulfide bond thiol groups.
Saving fluorescent probe as follows: 1) a fluorescent probe to be stored. 2) dry goods can be stored claroline for more than one year at -80 , such as unconditional, please stored at -20 . 3) It is strongly recommended to use RNase-free of TE (pH 8.0) buffer to dissolve the probe, the probe solution thus obtained is more stable and longer lasting. Typically, the probe is formulated as a stock solution 100 pmol / μl, and distributed into several claroline (up to repeated freezing and thawing five times each) and stored at -20 . Before use, the prepared stock solution was diluted to working solution (10 pmol / μl or 20 pmol / μl), the remainder of the stored at -20 . 37. What are the common fluorescent dye?
Full name abbreviation absorption wavelength emission wavelength color 6-FAM 6-carboxy-fluorescein 494 nm 518 nm Green TET 5-tetrachloro-fluorescein 521 nm 538 nm Orange HEX 5-hexachloro-fluorescein 535 nm 553 nm Pink TAMRA tetramethyl-6-carboxyrhodamine 560 nm 582 nm Rose ROX 6-carboxy-x-rhodamine claroline 587 nm 607 nm Red Cy3 Indodicarbocyanine 552 nm 570 nm Red Cy5 Indodicarbocyanine 643 nm 667 nm Violet
Dual-labeled fluorescent probe by the quencher TAMRA, Eclipse or BHQ series dye composition is often used as a hydrolysis probe (Hydrolysis Probes), also known as TaqMan claroline probe for real-time PCR experiments. 1) TAMRA fluorescent dye, quencher reporter group at the same time, will emit fluorescence at a higher wavelength. The Eclipse claroline and BHQ series when non-fluorescent dye, quencher groups report itself does not emit fluorescence, fluorescence in the background is lower than the TAMRA, higher detection sensitivity. 2) absorption spectrum coverage TAMRA narrow, relatively few can match the kind of reporter groups; while Eclipse is a wider absorption range (390 nm-625 nm), the report can be quenched many types of groups, such as FAM, HEX, TAMRA, ROX, etc. can; absorption spectrum coverage BHQ series of dyes used in combination are wider, has from 430 nm to near infrared, can be quenched more types of reporter groups, including Cy3, Cy5, etc. . So by Eclipse or BHQ dyes consisting of a series of dual-labeled fluorescent probes for multiplex PCR. 40. What are the basic principles of TaqMan probe design?
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