E coli culture cannot grow overnight. - (Jun/13/2014 )

Bio-Lad on Mon Jun 16 10:57:09 2014 said:

Oh! Ha ha. Here I thought I was still taking to the OP! Sorry about that! I've been sleep deprived lately due to our baby waking us up all night so I'm going to blame her for this! I was getting really confused. Lol.

What kind of total yield are you getting and what protocol are you using to get your plasmid, Ivok?

I get between 40 and 70 ng/µl. THis is too low (I think?).

I am using a sigma aldrich miniprep kit, just a commercial kit.

lyok on Tue Jun 17 20:33:27 2014 said:

 

Bio-Lad on Mon Jun 16 10:57:09 2014 said:

Oh! Ha ha. Here I thought I was still taking to the OP! Sorry about that! I've been sleep deprived lately due to our baby waking us up all night so I'm going to blame her for this! I was getting really confused. Lol.

What kind of total yield are you getting and what protocol are you using to get your plasmid, Ivok?

 

I get between 40 and 70 ng/µl. THis is too low (I think?).

 

I am using a sigma aldrich miniprep kit, just a commercial kit.

 

 

 

If eluted with 50ul (promega kit recommendation), that means you got 2-3.5ug total which actually isn't all that bad if you prepped only 1mL of that culture.  It's lower than what I'd expect if you prepped all 3mL.  If this is the case, I'd make sure you are getting complete lysis.  Occasionally if I have a big pellet, I will increase the amount of each buffer by 1.5-2x (resuspension, lysis, neutralization).  This usually helps with incomplete lysis.  I'd try that first if you haven't already.