difficulty with plasmid prep / restriction / gel extract

[marcusho]

I am having trouble constructing a plasmid from bacteria used to assemble shRNA lentiviral particles.

after isolating a plasmid from bacteria, and performing a double restriction digest, i am having trouble extracting a 7kb fragment that is released from the 9kb plasmid after digestion.

As seen on the attached gel pic, the 9kb and 7kb fragments do not separate very well and show up as a big blob. I tried running the gel longer for a better separation but did not work very well. I used qiagens’ plasmid purification midi and mini kits. Even loading small amounts per well (3uL) did not give a nice separation.

the plasmid is a pLKO.1 TRC cloning vector from addgene. Addgene included in their instructions to do the extraction using the qiaquick gel extraction kit. [Only registered users see links. ]

i loaded 6ug of DNA onto a 0.8% agarose gel using TBE buffer, 2hours at 125 volts. I also tried using TAE buffer, low mp agarose, and a different invivogen gel extraction kit as well but those didn't work.

During the extraction i used as little UV exposure as possible, kept it under 400mg per sample, made sure the agarose dissolved completely in buffer QC, heated the absolute ethanol PE buffer to 65 before using, and followed all "additional steps". I let the column stand 2min before eluting with water and before spinning down with PE. After eluting in 35uL dH2o, the yield is always < 30ng/ul. furthermore, the nanospectrophotometer reading shows a high peak at 225wavelength and very very little absorbance in 260.

what am i doing wrong? Why can’t I separate 7 and 9kb from each other, and why is there a crappy yield on the gel extraction?

Thank you

[mmorgan]

It's a little hard to tell from the images but it appears as though there is only one band at the top of the gel. 9kb and 7kb should separate without too much trouble. You say that you also tried running less DNA and you don't see two separate bands. I would be concerned that the plasmid is not correct. Maybe try cutting with some other enzymes to verify that the plasmid is exactly as it should be.

If the problem still seems to be that there are truly 2 bands that are not resolving, run a large (~30cm long) 0.7% agarose TAE at 60V overnight (without ethidium bromide). Then stain with EtBr the following day. You get better resolution when the gel is run without EtBr.

For gel purification I always use the Qiagen QIAEXII kit. The yield is not brilliant, but the DNA is good quality and I have used it to clone fragments as large as 20kb.

[hairongxu88]

tanks