I'm trying to purify 2-5 ug of my vector insert from a plasmid digest for injection into mouse embryos to make transgenic mice.
I'm digesting 10-20 ug of maxi prepped vector and running it on a non-EtBr stained gel. I then seperate one lane of the gel and stain with EtBr to show the band I want to cut and then line this up with the unstained gel and cut out the same band in the unstained gel. Following this I use a generic Gel extraction kit (Clontech's version) and dissolve the agarose + gel in chaotropic buffer (200 ul/100mg gel) and purify on a silica column. I am eluting the DNA in injection buffer (7.5mM Tris pH 7.4, 0.5 mM EDTA) and as my insert is ~3000 bp I am eluting the from the column by incubating column + buffer at 70oC for 5 min and repeating this three times with 20 ul of buffer everytime.
When I quantify the DNA concentration on a nanodrop, I get very little DNA back (perhaps 800 ng from a 20 ug plasmid digest, half of which is the DNA of my insert (50% by mass)). I am not sure where all the DNA from the digest is going! I understand that the process is not very efficient, but is
Hi Chris,
1.- Check your gel: buffer ratio, Most of the kits i have used recommended 1:1 volume of Binding Buffer to the gel slice (volume: weight).
2.- Try using elution buffer (not injection buffer) and the check the concentration. If this helps, then you can add injection buffer (this dilution would hopefully won't be too bad).
3.- Alternatively, you can use only 10 ul of injection buffer each time instead of 20 ul of buffer every time.
4.- Try to load your gel with heaps of digestion, therefore starting with a huge band.
I hope this helps!
Stain the gel after you cut it to be sure you really got the band. Try electroelution... Put the gel slice in a bit of dialysis tubing fill with some running buffer and tie off. Fill your agarose gel box with running buffer. Put the dialysis bag in and run the DNA out of the gel slice in to the bag. Collect and precipitate, then dissolve in your buffer of choice.
Check how many micrograms you loaded on the gel, normally we do not load 20 ug , is it?
What are your digestion conditions? Could you also try add a second restriction enzyme that will cut the plasmid and not your insert, and therefore enable you to discriminate the two bands more easily? That might give you more room to cut?
we use QIAGEN kit to purify plasmid DNA that works well for us
Hi Chris,
I also had the same issue with you. I started the digestion with 30 ug and I ended to a mass of 600 ng (70% of which is my interested gene insert).
I did a phenol extraction though that it was quite roughly forthe DNA quantity.
The purification was done by qbiogene and I split my sample in 2 filters thinking that i had more than 10ug (each filter can recovery up to 10 ug of DNA).
However, do you know what is the final concentration in the injection buffer? And how much volume is needed?
Thanks.
Speaking to other people in neighboring labs it seems quite a common phenomenon. These column gel extraction kits are inherently inefficient. From a 50ug digest (5 x 10ug - 50% of which is my DNA of interest) and purified on five columns I eluted by doing 3 x 5 min incubations at 70oC between spins. I ended up with 2.5-3.0 ug of plasmid at ~30 ng/ul. I diluted this down to ~250ul in sterile injection buffer to make 10 ng/ul. I ran 100ng on a gel to check purity and correct size. Currently being used to make mice.....
Stephen I was already cutting the vector DNA with two enzymes to make it more distinct from the required dna
In which types of columns had you done the purification?It sounds great, at least according the previous attempts, starting with 50 ug and resulting with ~3 ug. So, you did the whole procedure from the very starting point of digestion..
I was wandering if I could skip this and doing something with my already purified sample like precipitating it and cocentrate in smaller volume.
Just thoughts....
You could do that I guess. We use clontech's nucleospin pcr clean up/gel extraction kit. But any silica column kit should would the same really
We also encountered similar problems with transgene purification. We got best results of transgene purification (of linearized DNA) using a 5-20% sucrose gradient following recovery of the DNA fragment under UV light.
Further, we keep an appreciable size difference between the transgene and the vector backbone.
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