Sequencing (3-#39; End Sequencing, 5-#39; End Sequencing)

asked:
just do cloning, meet the sequencing process there are 3' end sequencing, 5' end sequencing, what is going on? Why are there two sequencing results? Should I follow that one to see if the sequencing is correct or not?
:
sequencing is carried out from the 5' end, forward and reverse sequencing refers to the two complementary chains of
DNA
are sequenced separately, usually the two directions of sequencing results after reading exactly the same can be considered reliable results. A bit of sequencing experience:
1, sequencing selection of quotations is very important, the general clone vector now has universal quotations, such as T7, SP6, M13-47, RVM, .... But which lead is chosen for sequencing? Our laboratory often sequences, and it has been proved that the position of the quotation in the carrier is preferably about 100bp upstream of the starting point of the target fragment, such as the pGEM-T carrier, if the positive (5' end quotation), can be used T7, M13-47 can also be used, but with T7 (more than 50 points from the insertion point), often appear the purpose sequence before dozens of bp can not measure the phenomenon, with m13-47 better, although will measure more carriers. (This is because dozens of bp after the quotation are untested, and the end-of-deoxygenation termination method is not measured).)
2, sequencing results of the proofreading: if the company sequencing, their own purpose sequence, sequencing purpose is to verify, according to the peak chart correction (the company generally does not do this work).
3, if you sequencing ourselves, we are using ABI 3100-Advant, sequencing system is very important, BigDye can be diluted, but Buffer must do the corresponding supplement. The final concentration of the quotation 0.15uM is very good, as if it
lower than
ordinary PCR concentration. The concentration and purity of the protons (or PCR products) are critical!! The prosurgers are best
in
box, and the concentration remains at this level: we have a 20ul system with a guarantee of 100-300ng.
asked:
sequencing report single came, trouble also came: how to see to know that there is OVERLAP? I'm dizzy. Also: 5' short sequencing results are not complementary to the
I
purpose? That is, consistent with the upstream lead sequence?
:
with specialized sequence analysis software, such as sequencher, DNAstar, etc. know that if you have overlap, the result should be 3' end of sequencing should be paired with the end of 5' end sequencing (how much of the pair depends on the length of a reaction you measured). Second, the 5' end sequencing results should be consistent with the upstream lead sequence, but your upstream lead sequence is generally not seen in the 5' end sequencing results, even if you see only a few bases.
.