Restriction Fragment (urgent) - (Jul/08/2007 )
Hello, I'm having problem on how to read restriction fragment after the enzyme digestion. I cut my plasmid with XMN1. This enzyme cut my vector (size 5753bp) at 287, 734, 1132, 3063 and cut once at my insert (insert size: 1317) at 374. The insert attached / goes in at 305 of my vector. Can someone be kind enough to teach me on how to read the expected restriction (so i identify any positive plasmid.. if any).
Since I'm really new in this area, maybe anyone can suggest to me on some reference -website / books / papers- (to know correct / reverse orientation) on restriction enzyme fragment.
Thanks in advance for your help.
For reference, try to use Sambrook and Russel (some molecular biology book).
Back to your question, is your plasmid contain your insert or is it just a parental vector?
If you cut your parental plasmid with that enzyme, you will have 4 different fragments.
Maybe you should tell us more about the plasmid you are using.
Hello, Thanks for your quick reply... The vector i use is pET 101 by Invitrogens. The plasmid is with the insert (plasmid + insert).
ok, if you have your insert there, then you must get 5 bands considering your plasmid is completely cut. band sizes should be: 392bp, 1372bp, 398bp, 1931bp, 2977bp. but i don't think you can separate 398bp and 392bp bands on agarose gel.
if you do not have an insert, then you will get 4 bands. sizes: 447bp, 398bp, 1931bp and 2977bp
do you need to check your positive clone with this restriction enzyme. I would try something that do not cut my vector but cuts insert. don't you have such an enzyme??
Hello, Thanks to Timjim and Dodosko for your reply, I would take both of advice in analysing my samples.
Thanks again guys!