Analyze the fourth for Composition and T m, will give the upstream primer composition ratio and Tm

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This window can be linked to "primer search", "primer Edit" and "Search Results" option, click the Search button, trypanosoma enter primer search box, select trypanosoma "PCR primers", "Pairs", set the search area and primer length and products length. In Search Parameters inside, you can set the appropriate parameters. General absence of special needs, you can choose the default parameters, but the product of the length can be appropriately changed since 100 ~ 200bp product electrophoresis run more scattered, so you can choose trypanosoma 300 ~ 500bp.
For primer sequences, trypanosoma you can simply look to avoid the following: 3 'do not appear in the case of three consecutive bases connected, such as GGG or CCC, otherwise easily lead to mismatches. It is important to view this window include: T m should be between 55 to 70 degrees, GC% should be between 45% to 55%, T m values of the upstream primer and downstream primer is best not too much difference, probably in two degrees better. The bottom of the window lists information about the secondary structure trypanosoma of the two primers, including, inter hairpin, dimer, primer cross-dimers and false priming position. If the button is red, indicating the presence of the secondary structure, click on the red button, you can see the position of the secondary structure of the corresponding icon. Ideal primer should not exist these two structures, namely these buttons are displayed as "None" as well. But sometimes it is difficult to find various conditions are met primers can be relaxed so requires, such as the presence of primer mismatch, you can look at how the mismatch efficiency, will significantly affect the product on the specific circumstances. Primers specific for detailed evaluation needs to be completed by means of Oligo, Oligo primer itself though trypanosoma with the search function, but its search for a quality feel than the primer Primer5.
In Oligo software interface, File menu, choose Open, navigate to the target cDNA sequence (in the primer, the sequence has been saved as a file Seq), pop up two windows were Internal Stability (Delta G) window trypanosoma and Tm window. In Tm window, click the lower left button, the primer will come out positioning dialog box, enter the location of the upstream primer sequences candidate (Primer5 been given) can be, and primer length by clicking the Change - Current oligo length to change. After pointing, clicking Tm window Upper button, determine the upstream primer, the same method of locating the position trypanosoma of the downstream primer, click the Lower button to determine the downstream primer. After the primer is determined, which can take advantage of a variety of powerful Analyze menu primer analysis function.
Analyze, the first term of Key info, click Selected primers, will give two primers trypanosoma general information, including the T m values of the primers, Oligo This value is calculated using nearest neighbor method, the primer will be in Tm than Primer5 value is slightly higher, this window also gives primer Delta G and the 3 'end of Delta G.3' end Delta G is too high, the site of the mismatch will form a duplex DNA structure and cause the polymerization reaction, and therefore the absolute value of this should be small, it is best not more than nine.
Analyze second term as Duplex Formation, namely dimer formation analysis, you can select trypanosoma an upstream or downstream primer primer dimer in the case of formation and downstream primer between the upstream primer dimers between, you can also choose to Upper / Lower, namely downstream primer dimer formation between trypanosoma the upper. Primer dimer is an important trypanosoma factor trypanosoma in anomalies affect the PCR reaction, and therefore should be designed to avoid the presence trypanosoma of primer dimers, at least make dimers formed by the designed primers are unstable, i.e. it Delta G value should be low, generally trypanosoma do not make more than 4.5kcal / mol, combined with not more than three base pairs. Oligo this analysis are given a window of the 3 'end and the entire primer dimers illustrations and Delta G value.
Analyze the fourth for Composition and T m, will give the upstream primer composition ratio and Tm value of each base primer and downstream products. GC% upstream and downstream primers need to be controlled in 40% to 60%, and the GC% not much difference trypanosoma between the downstream primer. There are three Tm values were calculated using three methods, including the nearest neighbor method,% GC method and 2 (A + T) + 4 (G + C) method, the last one should be the method used Primer5, T m value can be controlled between 50 to 70 degrees.
search out primers Sort by Rating, individually send Oligo software in evaluation. Of course, the search for the primers, an amplification trypanosoma product which is very short, you can not select it, or a primer 3 end 2 A or T, or internal primers were too many consecutive G or C, or a primer 3 end 2 G or C, and this should be used as a primer second choice, did not get elected to vote for it. For such a primer, if other indicators can, I like to remove the primer end a dissatisfied or add a base to see the primer evaluation parameters have not changed a good point.
In analyze where, Duplex Formation whether upstream primer, or the downstream primer between the upstream and downstream primers, The most stable 3'-Dimer absolute value should be less than 4.5kcal / mol, The most stable Dimer overall absolute value should generally be less than the number of kcal / mol with PCR annealing temperature, several experiments I feel at the time PCR annealing temperature of 65 , The most stable Dimer overall trypanosoma 6.7kcal / mol no problem.
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