Help restriction analysis of plasmid - (Jun/10/2008 )
I think it is the concatemers. Meaning, for your clone, you have 2 inserts ligated and thus they are ligated to the vector. That is the reason you have twice the size. It happens because you are using single digestion to cut at 2 positions. Try double digestion instead.
Just sequence it and you can find out about your insert. Good luck.
-timjim-
QUOTE (Sherry_Tong @ Jun 11 2008, 06:51 AM)
I am a novice in cloning reaction, and I am trying to clone gene of 1.1kb into pET-100/D-TOPO (invitrogen).
PCR was done by using AccuPrime Pfx super mix and primers designed and genomic DNA. After the clone reaction, I find several colonies on my plates (LB+Amp). I cultured the colonies from my plates overnight, and purified plasmid using purification kit.
To confirm the insert, restriction digestion was done by using Hind III. Hind III should cut both vector and insert once. So, I suppose to find one band of 5.6 kp and one band of 1.2 kp on the gel. But, I found one band of 5.6 kp and another band of 2.4 kp on my gel.
What could be wrong in my experiment? Thanks for any suggestion.
PCR was done by using AccuPrime Pfx super mix and primers designed and genomic DNA. After the clone reaction, I find several colonies on my plates (LB+Amp). I cultured the colonies from my plates overnight, and purified plasmid using purification kit.
To confirm the insert, restriction digestion was done by using Hind III. Hind III should cut both vector and insert once. So, I suppose to find one band of 5.6 kp and one band of 1.2 kp on the gel. But, I found one band of 5.6 kp and another band of 2.4 kp on my gel.
What could be wrong in my experiment? Thanks for any suggestion.
So have you checked the site on your insert? Maybe that's a theoretical site. So just make sure the site Hind III can be used for the digestion, means you'd better do a single restriction digestion by using HindIII with ur insert.
Good Luck
-vincenttianfr-