i m new to bioforum, it seems quite interesting. I am expecting i wud certainly get my solution here.
I am trying to clone a 6.8kb long gene in to pET21b(+) expression vector (size ~5.4kb). After a bit of struggle i have amplified full length of gene, i also incorporated BamHI site at 5' end and HindIII site at 3' end of gene to construct direction clone of gene. In order to detect the presence of gene I use a different set of primers which are highly specific (FP is 27 bp long and RP is 26bp long) and gives 927 bp long amplified product.
I have got a recombinant colony. I extracted the plasmid from recombinant and checked for the presence of gene by PCR using gene specific primers. I also kept +ve control (genomic DNA), -ve control (empty vector) and recombinant plasmid and got exactly 927bp long product from genomic DNA and recombinant plasmid but not from empty vector. upto here every thing looks ok but when i look into size of the plasmids it is having identical size as empty vector. since insert size is huge, it should certainly reflect in 1% agarose gel.
I have repeated PCR but again getting the similar result. I tried to see whether insert was overdigested, may be due to star activity of RE but found only one band in gel i.e. RE digestion before ligation step was perfect and it was ~6.8kb.
When i tried to release the insert by double digestion i saw a no of bands (~10 bands) which spun my head, i m totaly lost..i m nt able to understand how it is possible.!! some friends suggested that may be part of gene is inserted and giving 927bp fragment but again i sud be able to see this difference (1Kb) over agarose gel. 1 kb difference is easily detectable but recombinant plasmid looks ~5.4 Kb only even after digestion by BamH1 to release its superhelical structure.
Plz help me out
Looking forward for ur positive gesture.
thank u all in advance,
Edited by sks1, 21 October 2011 - 04:58 AM.