3 replies to this topic

[ChrissyFL]

Hi, I am a new grad student, just learning how to clone and I have been having numerous problems without the help of others..

I'm cutting a plasmid with EcoRI and it is not cutting very well.  I am using the 10x buffer that came with it but maybe I am using not enough..?  I'm not sure.  I asked the postdoc in our lab and she didn't seem to be 100% sure either   So... can someone please explain to me (like I'm a kindergardener!) how much of a 10x buffer I should use?  I am under the impression that you use 10% of the final reaction volume.  For a 20 ul reaction, you would use 2 ul of 10x buffer.  For a 50 ul reaction, you would use 5 ul of 10x buffer.  Is this correct?  Does it matter how much DNA you are using?

Thanks!

[hobglobin]

ChrissyFL, on Mar 14 2009, 08:17 PM, said:

Hi, I am a new grad student, just learning how to clone and I have been having numerous problems without the help of others..

I'm cutting a plasmid with EcoRI and it is not cutting very well.  I am using the 10x buffer that came with it but maybe I am using not enough..?  I'm not sure.  I asked the postdoc in our lab and she didn't seem to be 100% sure either   So... can someone please explain to me (like I'm a kindergardener!) how much of a 10x buffer I should use?  I am under the impression that you use 10% of the final reaction volume.  For a 20 ul reaction, you would use 2 ul of 10x buffer.  For a 50 ul reaction, you would use 5 ul of 10x buffer.  Is this correct?  Does it matter how much DNA you are using?

Thanks!

The provided buffer is 10x (or 10-fold) concentrated. Dilute it with your final reaction volume to 1x buffer.

[phage434]

You have it right.  A typical reaction would digest 1 ug of DNA in a 50 ul volume like this:

xx ul    DNA sufficient for 1 ug
  5 ul    10x buffer
0.5 ul   100x BSA (not necessary for some enzymes)
   1 ul    enzyme
yy ul     water (sufficient to make the final volume 50 ul)

Tap to mix, spin briefly to bring it to the bottom of the tube.
Digest for 1 hour at 37 (or possibly higher, depending on the enzyme)
Heat kill the enzyme for 20 minutes at 65 (possibly higher) for 20 minutes
   (some enyzmes cannot be heat killed)

It's best if there is a relatively low volume of DNA, since then the reaction setup dilutes possible contaminants in the DNA solution.

[ChrissyFL]

Thanks so much!  You've just put my mind at ease!