Restriction analysis of a vector - (Oct/03/2005 )

hi, HomeBrew,
I just saw a single band after digestion but three bands before digestion,so the plasmid should be linearized by digestion. By the way,the plasmid (no cloning reaction,just vector alone) was provided by others and he insisted that it was right

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Was it a strong band or very faint ? If it's faint then maybe you just miss the insert band ! 60ng is really too few to visualize a small insert !

pesji

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Three bands before digestion is correct, but your double digests aren't (as you undoubtedly know...).

Double digesting pET32a (vector only, no insert) with EcoRI and XbaI should produce a 5363 bp band and a 537 band, while digesting with EcoRI and PstI should produce a 4568 bp band and a 1332 band.

So, here're are a few questions, and a couple of things to think about trying:

• Have you cut the plasmid with each of these enzymes singly and seen just a 5.9 kb band?
• If you run out at EcoRI + XbaI digest and the EcoRI + PstI digest in adjacent lanes, is the single band you can see in each lane the same size?
• What size is the single band you are seeing?
• The pET32 series is unique in that they have the Trx-Tag. Can you detect the presence of this tag by PCR?
• The Trx-Tag also contains a unique site for RsrII. Does RsrII linearize the plasmid?

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Homebrew, thank you for your good suggestions!
I can see the single band and the same size (about 5.9 kb ) in each lane after I ran out at EcoRI + XbaI digest and the EcoRI + PstI digest in adjacent lanes. But I have not cut the plasmid with each of these enzymes singly.
I can not detect the presence of Trx-Tag by PCR,so I have no idea about the experiments now.

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You can't detect the Trx-tag as in "I don't have the ability to" or you can't as in "I tried but it's not there"?

If it's the latter, someone sent you the wrong vector...

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Thank you,HomeBrew!

As I tried but couldn't detect the Trx-tag by PCR, I think that the vector someone sent me is wrong, and fortunately, I have got this vector from others and the results are OK.

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You know, some times the data is just telling you something right from the start that sounds so unlikely, you just refuse to believe it...

Don't ask that first lab to send you any more vectors!

Glad you got it sorted out!

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Hi,

I want to get some information about pCAGGS plasmid.As far as I know it contains chicken beta actin promoter and an cmv enhancer.I am trying to cut EYFP gene from pEYFP plasmid and ligate it to pCAGGS to get brighter EYFP.But ligation-transformation procedure always fails!!:(its MSC is very short and so restriction sites are too close to each other (6-7 base)

if there is somebody that has studied with this plasmid before or know something about it, could you please share your experience with me ?

Thanks in advance.

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