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problem in gene cloning


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4 replies to this topic

#1 novagen

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Posted 16 August 2009 - 08:03 AM

HI ALL,

I am cloning 800bp fragment (amplified using gene speific primers) into PTZ57R/T vector,transformed into E.coli,did plasmid isolation then restriction digestion of the plasmid. surprisingly my insert size is around 1Kb. how could this happen?
Iam very much sure i did not contaminate the sample. becoz I have checked the sample after gel elution, it was 800bp only.
and shockingly my primer set is amplifying 1kb fragment when I have used plasmid DNA as template for PCR. unable to untangle the problem.
suggestions appreciated.

ThanX in advance
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#2 perneseblue

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Posted 16 August 2009 - 09:35 AM

the quickest thing to do would be to
1 - sequence your DNA insert to make sure it is the right thing.
2 - digest your plasmid + insert with a different set of restriction enzymes, something that would cut your insert more than once would be nice.
2- do you have another isolate of your plasmid? Look at 3 others. Are they all the same? Could this one isolate simply be an oddity.
May your PCR products be long, your protocols short and your boss on holiday

#3 phage434

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Posted 16 August 2009 - 09:36 AM

How are you determining the insert size? Restriction digest of the prep's DNA? PCR of the plasmid? With what primers? Have you run your PCR product and your insert on adjacent lanes in a gel?

#4 novagen

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Posted 24 October 2009 - 07:24 PM

Hi all,

I did all the 3 things you mentioned.
Actually my original PCR product (800bp)and colony pcr amplified product (1kb) are not same.
they differ in molecular wts. and somehow i got sequenced the plasmid and did blast against NCBI db.fortunately or unfortunately it showed 100% homology with a bacterial gene but my source was a plant gene.
how do i go about with this.
now I feel I have to clarify wheather that gene was from my source or a spurious amplification. If it was a spurious amplification my primer set should not amplify the product but iam getting 1kb band with that primers.
how to untangle this problem.

thanx in advance
Service to man is service to God

#5 perneseblue

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Posted 24 October 2009 - 09:09 PM

hmm... do your primers have sequence homology to this bacterial gene?


You could try the PCR again and see if you do get the desired 1000bp product.
May your PCR products be long, your protocols short and your boss on holiday




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