Positive and negative controls for transformation - What controls to use (Apr/27/2006 )
Hello there,
This is my first time doing transformation and I am confused as to what kind of controls to use. Everyone keeps telling me it is best to use several controls, as well as a positive and negative control but I'm not sure which is which. These are the controls that was suggested:
1) Uncut vector alone
2) Cut vector alone
3) Cut vector + ligase
4) Cells alone w/o DNA (to make sure my competent cells are working I guess?)
So which is my positive control and which is my negative (the cells alone?)? What are these supposed to show and what should I see on my plates if they were working correctly? I thought for controls 1-3 on the list they should show the same thing, that is, lots of growth. What's the difference??? Am I supposed to see similar amount of growth on my positive control and my insert+vector plates?
Hi,
1) Uncut vector alone --> shows if transformation works. If so: many colonies
2) Cut vector alone --> without ligation you should see no colonies. If you see them, then your vector was not completely cut (a certain percentage)
3) Cut vector + ligase --> after ligation of cut vector (without insert) you should see colonies. Then your ligation works in principle.
4) Cells alone w/o DNA (to make sure my competent cells are working I guess?) --> No! Cells alone don`t grow on LB Amp because they don`t have the resistance gene located only on the vector.
I would say 2) and 4) are neg. controls.
Competent cells are able to take up circular DNA. If you transform them with a plasmid that contains a resistance gene for Amp or Kan or Neo or whatever, then only those cells which took up a plasmid can grow on plates containing antibiotics.
You can do 5) cut plasmid + cut insert --> ligate and transform.
With the help of your controls you can evaluate your transformation result.
1) Uncut vector alone --> shows if transformation works. If so: many colonies
2) Cut vector alone --> without ligation you should see no colonies. If you see them, then your vector was not completely cut (a certain percentage)
3) Cut vector + ligase --> after ligation of cut vector (without insert) you should see colonies. Then your ligation works in principle.
4) Cells alone w/o DNA (to make sure my competent cells are working I guess?) --> No! Cells alone don`t grow on LB Amp because they don`t have the resistance gene located only on the vector.
I would say 2) and 4) are neg. controls.
Competent cells are able to take up circular DNA. If you transform them with a plasmid that contains a resistance gene for Amp or Kan or Neo or whatever, then only those cells which took up a plasmid can grow on plates containing antibiotics.
You can do 5) cut plasmid + cut insert --> ligate and transform.
With the help of your controls you can evaluate your transformation result.
My boss says 2) is to check how efficient my gel purification of my vector was, and to check on the background? whatever that means....
hi
you're right. 2 is a negative control as it tells only how much uncutted vector remains in your prep "vector purified".
i consider as the truth negative control the vector alone + ligation.
Cells only are to check for plasmid contamination of your cells and for your cuvette / LB and check for selection eficiency too.
Thanks it's much clearer now!