problem in gene cloning
Posted 16 August 2009 - 08:03 AM
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.
ThanX in advance
Posted 16 August 2009 - 09:35 AM
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.
Posted 16 August 2009 - 09:36 AM
Posted 24 October 2009 - 07:24 PM
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
Posted 24 October 2009 - 09:09 PM
You could try the PCR again and see if you do get the desired 1000bp product.