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Tips and protocols for preparing DNA

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  • Digest at least 10ug (but up to 50ug) of maxiprep plasmid with suitable enzymes (for at least 2 hours).
  • Check digest (run a small amount on to gel to check it is cut properly).
  • Run on 0.7%  agarose gel (conc of gel is kept low so that agarose contamination is minimised, for the same reason it is good if the gel is overloaded).
  • Cut out the required band and purify with Qiagen gel purification system.
  • Elute DNA with 60 ul transgenic injection buffer (5-10mM Tris-HCl pH 7.4, 0.1-0.25mM EDTA – normal TE is not suitable).

To calculate concentration of DNA for injection (it is important that DNA be very accurately quantitated):

  • Using a square of plastic (say 10cm x 10cm cut from a clear plastic bag) aliquot out 1µl drops of standard concentrations of lambda DNA i.e. 25, 20, 15, 10, 7.5, 5, 2.5, 1, 0ng/µl, across the film.  Then place 1µl drop of DNA for injection as well as a 1:10 dilution of DNA.  Add 1-2 µl of ethidium bromide (10mg/ml) to 10 mls water and then add 1µl of this mix to each of the drops on the film.
  • Sit the plastic on an injection slide for easy transport to transilluminator.
  • View the drops by placing the plastic onto a transilluminator and measuring the DNA concentrations from the standards.
  • Give concentrated DNA to TASQ for injection (do not freeze diluted DNA). Best if diluted just before microinjection.

It is absolutely crucial that DNA be very pure for injection – ie free of phenol, alcohol, agarose, enzymes etc – helps keep injected embryos viable. It is also important that DNA be free of particulate matter that could clog injection pipettes – so usually spin for extended period (eg 30 mins) just before injection.

There are at least 4 methods for producing transgenic mice using BAC DNA.

Our preferred method is to microinject Pulse Field Gel Electrophoresis (PFGE) – purified BAC insert. PFGE is used because DNA 15-20kb or larger in size will migrate with the same mobility regardless of its size during continuous field electrophoresis. Having an alternating gradient voltage will give better resolution of these larger molecules. This procedure takes longer than normal gel electrophoresis due to the size of the fragments being resolved and the fact that the DNA does not move in a straight line through the gel.

However, we know that not many people have access to or experience with this kind of equipment and therefore cannot give us PFGE purified BAC DNA.

We recommend using the QIAGEN Large-Construct Kit (Catalogue number 12462), which is for the purification of up to 50μg BAC, PAC, and P1 DNA or up to 200μg cosmid DNA, free of genomic DNA.

Cost is $585.90 for 10 preparations.

 

The above is combined with the last steps of YAC (Yeast Artificial Chromosomes) DNA purification method, which is essentially dialysis of the BAC DNA preparation on a floating 0.05um Millipore Membrane against an excess of 50-100 times microinjection buffer with polyamines for a couple of hours.

At this point a 260/280 ratio will be relevant as the dialysis will have removed a lot of the contaminants from the sample. On its own, the QIAGEN prep will not be clean enough for microinjection and a 260/280 ratio will not be relevant as the sample will have contaminant agents in it which will corrupt your spectral reading.

A few pointers:

  • DO NOT ethanol precipitate. Treat with phenol or freeze down. Please store at 4°C.
  • The preparation should be crystal clear. If you can see stuff in solution we will not inject it. You will have to do dialysis.
  • 260/280 ratio usually won't be meaningful and is pretty much useless with BAC DNA preps unless you dialise first since the standard BAC DNA procedure (i.e. QIAGEN columns) usually produce a DNA prep with contaminating agents that corrupt your spectral reading.
  • To properly measure DNA concentration of BAC DNA preps the best solution is using a fluorometer. Spectrophotometers or related apparatus (i.e. nanodrop) are less efficient since they read a number of other stuff in the usually dirty BAC DNA preps and therefore the concentration is nearly always wrong. Fluorometer reads in a 90 degree angle on glass cuvetters and upon reaction with a fluorofor which only reacts with dsDNA, no matter what else is in solution.
  • An even better estimation can be obtained by comparing your prep with a previous prep for which you know the correct concentration and you compare the EtBr intensities on a standard gel.
  • If you can't precipitate how do you concentrate your usually low amount of BAC DNA prep? Will you need to concentrate it? DNA could be dropped down to 0.1ng/μl and still be reasonably possible to get several transgenic lines. Or you can use Centricon microtubes to spin and concentrate samples.

 

References

There are 2 websites we recommend visiting before you start to get a feel for what is involved and any alternative methods that might suit you better. Please contact us before you submit DNA to us.

  1. Thom Saunder's Transgenic Facility web page at the University of Michigan where you'll find detailed protocols and comments on the preparation of BAC DNA for microinjections.
  2. Lluis Montoliu’s Protocols webpage at the National Centre of Biotechnology, Madrid, which has detailed protocols.