Is PX459 a high copy or a low copy plasmid?

[ZQ]

Hi everyone,

This is already the second time that I did miniprep but got only around 40-50ng/ul of my sgRNA constructs of PX459(pSp-Cas9-BB-2A-Puro). Is this normal? Normally I always get 300-600ng/ul from other vectors like pcDNA3.1, topo, ect.

Thanks in advance!

ZQ

[Anshuman Das]

Hi. its low copy compared to pcDNA. You need to do midiprep (50-100ml culture). So looks like you successfully cloned into px459 vector. I am having trouble in cloning into px334 vector. I am getting lots of self ligation colonies, almost same number in ligation plate. So which protocol did you follow?

Thanks
AD

[Phil Abbosh]

i was having the same problem with "no-insert" clones til i gel-purified the BbsI-digested plasmid.  there was actually an uncut band above the restricted band which was probably giving me all the empty clones.  i found out later that i was storing BbsI improperly (the package insert says -80C, i just put it at -20C with the rest without even thinking about it).  i never tried the golden gate protocol (simulatneous ligation/digest) because my ligase seems to work best overnight at 16C.

i did not kinase my inserts, and did not CIP the vector (I am using pX330 and pX335).  I diluted the annealed oligo approximately the way the Zhang lab protocol recommends (1:10 then 1:200) and then did a range of insert to backbone ratios (1uL of diluted oligo seemed to work best). Even with doing all of that, i still have occasional empty vectors but 11 out of 12 different oligos are properly inserted now.

[ZQ]

Hi Anschuman,

Please check your BbsI is cutting. And also I made a mistake because I was in a hurry ordering my botom oligos 3'--5'. That's why I also got a lots of self ligated colonies. Please check your primers just in case.

Now I got right clones with the right primers!  and both Zhang's proper protocol and dirty quick protocl worked fine!

Good luck!