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what primers should I use for DNA sequencing??


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#1 springstreet

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Posted 19 October 2009 - 05:41 AM

I only know the vector is pED-3d, but dunno the insertion site. Now I need to submit samples for DNA sequencing, please let me know what primers should I use? (T7? or something else) If I have to make primers myself (without knowing the insertion sites), how should I do it?

Thank you all so so much!!

#2 mdfenko

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Posted 19 October 2009 - 08:40 AM

the insert will normally be in the multiple cloning site (mcs). since you don't know any more then you should use a standard primer (like t7) if it is in the plasmid. try one that is about 100 bases away from the mcs.
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#3 springstreet

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Posted 19 October 2009 - 02:08 PM

the insert will normally be in the multiple cloning site (mcs). since you don't know any more then you should use a standard primer (like t7) if it is in the plasmid. try one that is about 100 bases away from the mcs.


thanks a lot for ur reply!!

#4 swanny

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Posted 19 October 2009 - 02:38 PM

vectorpedia has an entry for a mammalian vector pED (~5.4 kb; amp resistant), but nothing for pED-3d. Could it be pET-3d (4.6 kb, amp)??? Do you have a size for the vector? Try some standard digestions to look for unique sites - EcoRI, BamHI, HindIII. You may even pull out your insert...
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