confirmation of insert in to vector - (Oct/21/2011 )

Hi all,
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,

SKS1

PCR amplification of the insert only is unreliable. You should trust your gels, or do a pcr reaction with one primer on the plasmid an a second one on your insert, to eliminate amplification of insert-only DNA. I don't know why your digestion shows so many bands. You should digest the parent plasmid to check that the restriction enzyme(s) you are using cut only once.

phage434 on Fri Oct 21 12:48:54 2011 said:

Thanks for ur insight .. i get the feeling that using cross primers, one on plasmid and other on insert is gd idea, I'll try this!!. PCR is nt always very reliable but as i said primers are very specific and its giving exactly anticipated product so it is very hard to ignore bt ofcourse i cannot ignore gel observation too. i have checked for possibility of RE sites in insert as well as vector using NEBcutter ... there was no such site in insert ..so this possibility is eliminated.

can you plz suggest any of possible explanations of my observation?

The failure mode for insert-only pcr analysis of cloned samples is that the insert DNA comes through the ligation and transformation reaction and is picked up when doing the PCR of the colony. Remember that PCR can detect ridiculously small numbers of molecules of the target.