3 replies to this topic

[zhm]

I'm reserching in gene subclone.I know it must be very difficult to sucess in using single restriction endonuclease which cause blunt end to cut vecter and aim DNA ,and ligeasing them.
I have done the experment for about 6 month,but the there are no result . Can anyone give me some advice

[postdoc]

Hi ZHM

did you treat your vector with Calf Intestinal Phosphatase (CIP)?

The following may help

Plasmid Vector Preparation

Restriction digest and Calf Intestinal Phosphatase (CIP) reaction - 4-6 hours or overnight
Gel electrophoresis
Fragment elution and purification

Insert Preparation

Restriction digest -4-6 hours or overnight
Gel electrophoresis
Fragment elution and purification

Ligation Reaction

[dld2002]

Give us more information. What exactly are you doing? List all of your steps. Are you running your digested vector and insert on a gel to make sure that your restriction enzyme is cutting them?

[ale]

zhm, on Dec 13 2003, 06:52 AM, said:

 

I'm reserching in  gene subclone.I know it must be very difficult to sucess in using single restriction endonuclease which cause blunt end to cut vecter and  aim DNA  ,and ligeasing them. 
I have done the experment for about 6 month,but the there are no  result . Can anyone give me some advice

Hi.
Although it is not clear what is your problem, if it is about blunt end cloning into a vector, I suggest you:
1. If you can not chang the vector, use larger amounts of the insert, also, be sure of the purity of both DNA fragments to ligate, vector and fragment when using T4 DNA ligase.
2. I've got excellent results using Blunt end cloning from Invitrogen with TOPOISOMERASE strategy (serch for TOPO vectors in the web site according to the size of your insert).
Good luck!