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Why was one part of insert missing? - (Aug/11/2008 )

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A diagram would help, but if I understand you correctly, the problem is that you *never* get sequence results within or close to the primer used for priming the sequencing reaction. Sequence results start several bases 3' of the primer location, and give poor to very poor sequence for the first 20 bp or so. Good sequence starts around 30 bp after the end of the primer, except for dye blobs, which occur about 80 bp and 120 bp into the sequence results. You don't always see dye blobs, especially if the sequencing reaction is optimized (with the correct amount of template DNA and good cleanup). If you need sequence at the very beginning of a PCR product, you have to sequence in the opposite direction with a primer that is close enough to the end (within 500-800 bp, depending on how good your sequencer and sequencing reaction are).

-phage434-

Hi!

Here are some figures thtat can explain the problem, phage434; attached is the Power Point. As you can see, the primer used to sequence is about 70 bp upstream from the missing sequence, and the most of the bases (end of the GFP sequence) before the 20 bp fragment which we lack for, are correctly shown.

Lately, we've realized that there's a BciVI site (shown in the schemes) at the 20 bp we look for. The digestion of the pLenti construction was done, and the restriction pattern showed that enzyme cuts, but the 20 bp are not there. In the other hand, there's an EcoRI site immediately before the BciVI one. So, we amplified a 1.3 kb fragment including almost all the GFP, and the complete Fig insert. The EcoRI digestion was convincing: produces the two expected fragments, while the BciVI reaction lets an intact band. The lack of those 20 bp at the beginning of our insert has been confirmed both by sequencing, and restriction. What do you think guys?

We're guessing that if we lack of those 20 bp that include the 16 bp of the 5' primer for the first cloning, why our PCRs are still amplifyng the expected 585 bp fragment? The negative controls (no DNA into PCR reaction) are clean.

We need the complete insert (or at least, fix it to let it into a correct ORF). So, if we clone again, how can we avoid loosing its 5' end? It has happened twice in two different vector systems.

Thanks, so much!

Regards,

-spilo-

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