4 replies to this topic

[netnus]

hi,everyone,

  I recently isolate some plasmids which I don't know whether the inserts have been ligated or not! Before I send it to be sequenced, I want to know whether there is something ligated into the plamsid vector.
  Is there any quick way to determine the plasmid? For the insert is only about 400bp,but the vector is about 4300bp.So I can't unambiguously see the difference while I try to compare the ligated and vector even I cut them into linear.
  Can anyone give me some suggestion! I will be grateful for your help!



  netnus

[biomed]

netnus, on Oct 22 2004, 11:15 AM, said:

hi,everyone,

  I recently isolate some plasmids which I don't know whether the inserts have been ligated or not! Before I send it to be sequenced, I want to know whether there is something ligated into the plamsid vector.
  Is there any quick way to determine the plasmid? For the insert is only about 400bp,but the vector is about 4300bp.So I can't unambiguously see the difference while I try to compare the ligated and vector even I cut them into linear.
  Can anyone give me some suggestion! I will be grateful for your help!



  netnus

Hi, I am wondering how you made this clone?
1. Why do not check it with PCR for the insert?
2. In another way, if you can cut off the insert, 400 bp band will show up on the 2% argarose gel.
good luck.
Biomed

[postdoc]

Hi,

I think you can simply run a gel after miniprep to tell if a plasmid has insert. I have used vector of 4kb with 200 bp insert. I ran a 1.5% gel and could clearly see a difference between plasmid with and without the insert.

Edited by postdoc, 23 October 2004 - 12:48 AM.

[chandima]

hi,
may be if u first do a restriction digetion, decide the En, so that u get distinguishable sizes and run on a gell
good luck

[postdoc]

Yes, chandima is right.

Include a postive and negative control when running the gel.