Hi every one
I am working on PeT 28a for gene cloning. Since I use Sal I and NdeI for digestion, with electrporesis we can not recognize that it has been cut ( because it produces one small fragment and one big fragment). dose anyone know how can I recognise my vector has been digested or not?
Thanks in advance
0
2 replies to this topic
#2
leelee
-
- Active Members
-
- 631 posts
Veteran
49
Excellent
Excellent
Posted 02 September 2012 - 11:00 PM
Do you just want to check if your enzymes are working?
I would do a single digest with each enzyme on its own in addition to your double digest- and if they work, linearising your plasmid, then it is a good indication that your double digest will have also.
Will you then be doing a ligation with an insert?
Because if you then do a vector only control, you will get an idea of your background level of uncut plasmid from that (of course this will only tell you if one or other or both enzymes cut- but you won't be able to tell which).
That's all I can think of
I would do a single digest with each enzyme on its own in addition to your double digest- and if they work, linearising your plasmid, then it is a good indication that your double digest will have also.
Will you then be doing a ligation with an insert?
Because if you then do a vector only control, you will get an idea of your background level of uncut plasmid from that (of course this will only tell you if one or other or both enzymes cut- but you won't be able to tell which).
That's all I can think of
#3
phage434
-
- Global Moderators
-
- 1,805 posts
Veteran
130
Excellent
Excellent
Posted 03 September 2012 - 12:38 PM
Agree with Leelee. You can run an undigested lane on the gel, which will clearly show whether each enzyme digestion is working. Warning. If you are trying to cut and insert a PCR product, the SalI digestion of PCR products is known to have difficulties. Make sure you have many bases of 5' overhang on your SalI containing primer.
