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#1 2014-10-23 18:28:17

vithya
Member
Registered: 2014-10-23
Posts: 4

empty vector

Hi,
I used rfcloning to insert four fragments including promoter to modified PBR322 vector.
At each step the insert was confirmed with colony PCR. Everything was fine.
Now I sequenced the plasmid and I could find only the parent vector.
I doubt the Dpn1 digestion step and I want to repeat the procedure.
I would like to ensure the primer designing is correct.
The project ids

ef07449f1b3d5033ea240b3527b4ffef
bc4d8dc5b02356491553fbb5db1545d4
fdf694539142f5f415e48286d0b4010c

Vidhya

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#2 2014-10-23 18:45:23

Steve Bond
Administrator
Registered: 2014-01-23
Posts: 126

Re: empty vector

Hi Vidhya,
Sorry to hear you're having trouble. Your projects look fairly straight forward to me, and it's strange that your colony PCR looks fine if there isn't an insert. Can you elaborate a little more on what you did to confirm the insert by PCR? Maybe post a gel?
Fortunately, you do have a really handy EcoRI site inside your NVD trailer sequence, so you should try doing a diagnostic digest on a couple clones from that project. It will save you sending things off for sequencing that are obviously negative.
One other thing, you are definitely aware that you're breaking the Amp resistance gene, right? Are you selecting on Tet plates?
-Steve

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