Plasmid DNA purification

[ifhmn]

I have cloned my gene of interest using competent cells from qiagen and manage to get blue colonies. Then I inoculate 1 single colony and culture in the LB broth with kanamycin and grew overnight with shaking. I've checked my inoculum by running PCR and gel electrophoresis suing M13 primer and got very bright bands with the right size. Then I try to purify my plasmid DNA using Qiagen miniprep. However I got very low concentration (around 13ng/uL). I wonder how. And I have tried repeated it twice but still got low concentration. Does anyone got an idea?

[relaxin]

What is the plasmid you are using? It could be a low-copy plasmid. You can always scale up your miniprep to midiprep to get enough plasmid DNA you need.

Did you say you pick blue colony and get insert of correct size by PCR?

[ifhmn]

yes, I only pick the blue colony. I'm using pUC vector (from Qiagen cloning kit) that has high copy. How can I upgrade my miniprep into midiprep?

[ifhmn]

I've attached the picture of my PCR product (256bp). I only chooce the cultures that only showed 1 band

[relaxin]

pUC vector with small insert may give light blue colonies instead of white ones.

If you are using 5 ml overnight culture with the Miniprep kit, you can increase the overnight culture to 10 ml. But you have to double the volumes of Buffer P1, P2 and N3 during cell lysis. After clearing the lysate, then apply the entire supernatant to the spin column (since the lysate is more than 700 ul, you need to load the column twice). After washing with Buffer PE, elute DNA with 50 ul of Buffer EB.

[tluebke]

What are the amplification products with a size about 100 bp? I do not think that this bright band is just due to failed annaeling. This looks like a correct amplified product, as seen in the other samples. Are you sure that you do not have a mixed culture?

[talkingtree]

Sometimes I reload the eluted volume to the column for more yield. If you want more plasmids you should use bigger preps