wrong insert? right insert?? - (Nov/04/2013 )
Hi. I got a question related to insert size. I run my samples on agarose gel after restriction digestion. Some of the samples have insert but it looked slightly smaller than desired PCR product!!
However, my seniors told me the gel picture look alright and I should send my samples for sequencing. I really wonder are those correct inserts??? What do you guy think?
Following is my gel picture.
lane 1: 100bp dna ladder
lane 2: uncut plasmid dna (control)
lane 3: pcr product (control)
lane 4 to 23: samples
lane 24: pcr product (control)
lane 25: uncut plasmid dna (control)
lane 26: 100bp dna ladder
I think there is a good chance that you have the correct insert. Bands on gels run at different speed depending on salt concentration. Probably your insert DNA and restricted plasmid DNA are in quite different salt containing buffers. Another possible explanation could be that your insert has an unexpected restriction site for the enzyme that you are cutting, but you will find that out if you sequence. If you cloned with the same enzymes as you are cutting with now, this couldn't (in theory) happen.