How to identify plasmid - using enzyme and PCR or other? (Jun/02/2004 )
Now I have constructed one expression plasmid but it is one kind of low
copies. If using PCR amplification, only weak bands can be detected,
but if I use restriction enzyme to cut the plasmid, I can not see any target
bands to be cut after running agrose glue.
Why? And I wander if there are other better methods to identify my
plasmid, I hope someone can help me solve this problem.
Thank a lot !
Expression vector plasmids are many times low in copy number. If you cannot see the product after digestion due to low starting material, try cutting the plasmid at one site, preferably within the insert and in a separate reaction, at one site within the vector. The linearized plasmid sizes should match with each other and should be larger to the vector by a proportion equal to the insert size. At all costs avoid enzymes with star activity for this analysis or you are going to be pretty confused. Also, use the low melting LMP agarose at a strength of 0.8% embedded with 0.1% NuSieve GTG agarose. The gel should carry ethidium bromide in addition to the buffer so DNA bands can pick up the dye as they roll along
Thank you, phdconsult!
I will have a try at once. Wait for my good news!