7 replies to this topic

[resmi]

HI,

I am working on pHELLSGATE12 vector for RNAi in plants.  Initially I clone my gene in pDONR221 vector and LR recombined into pHELLSGATE12 vector.  I got numerous colonies on spectinomycin plate which on colony PCR and sequencing confirms the presence of insert (Using gene specific primer I got gene preceeding some regions of (approximately 300 bp) pDONR221?????). But on sequencing with pHELLSGATE specific primer (CaMV and OCS) I got approximately 800 bp reading in both orientation which is of the pEHLLSGATE vector region only. So whats happening.  I was confused????????  Does anybody know the solution.........  If a single colony contain more than one plasmid how should I proceed...

[Ameya P]

resmi, on 27 October 2010 - 08:53 PM, said:

But on sequencing with pHELLSGATE specific primer (CaMV and OCS) I got approximately 800 bp reading in both orientation which is of the pEHLLSGATE vector region only. So whats happening.  I was confused????????  Does anybody know the solution.........  If a single colony contain more than one plasmid how should I proceed...

It would be nice if you could confirm that the sequencing primer site was close to the actual insert (less than 800bp). As far as I know, sequencing reads are usually 800 bp and if you primer site is too far, you will just get the vector sequence and no insert sequence (even though the insert is present). I did not quite understand your 'more than one plasmid question'. Do you think that you have a colony with a realigned vector and a vector with the insert???? Kindly clarify.

[resmi]

I had done the gateway cloning using entry and destination vector.And after LR recombination i had sequenced the transformed clone (????)with the primer which should read the insert.  But I couldn't get the read (insert region) even after 750 bp (only the vector region). But there is insert in the colony which I confirmed by gene specific primer which is an enigma for me.  Then why should I doesn't got the insert using vector specific primer (primer very near the recombination site).

[HomeBrew]

If you're asking whether a single cell of E. coli can contain two different clones based on the same vector -- one clone with an insert and one without, in this case -- the answer is no.  Since the vectors are the same, they belong to the same incompatibility group, and the cell cannot maintain more than one clone.  There will, of course, be more than one copy of the unique clone in the cell; how many copies exist in a single cell is determined by the vector's copy number.

[resmi]

HomeBrew, on 29 October 2010 - 03:36 AM, said:

If you're asking whether a single cell of E. coli can contain two different clones based on the same vector -- one clone with an insert and one without, in this case -- the answer is no.  Since the vectors are the same, they belong to the same incompatibility group, and the cell cannot maintain more than one clone.  There will, of course, be more than one copy of the unique clone in the cell; how many copies exist in a single cell is determined by the vector's copy number.

[resmi]

If I inoculate from an E.coli colony and isolate plasmid then is there any chance that i got vector alone and insert relying on another vector?????????

[pDNA]

If you inoculate your colony PCR from the original ligation plate there is the possibility that you get a false negative signal due to traces of the original vector that was spread with the ligation reaction. You can streak-out your clones on a fresh plate and try to screen again ...you will see that this false negativ signal will go away!

Regards,
p

[Ragu]

Hi Resmi,
Did you find the answer?? I have a question.
Is your HG12 is called pStargate?? If so there might be a problem with your construct.
Try to match/blast the sequence result against your vector sequence and identify where it is amplifying. You will get an idea.
In my experience, the CSIRO RNAi vectors has some kind of problems like intron reversal etc.