Preparing Vector DNA For Transfection/Targeting

 

Since most people have their own preferences with this task you are completely at your leisure to ignore this section. This procedure works for me. However, be aware that highly purified DNA is important for high rates of targeting and transfection, so it is recommended that you at least peruse the sections below to obtain an idea of the level of purity required.

Plasmid Prep

-Most plasmids are kept in bacteria as glycerol stocks, restreak some out to achieve single colonies. If using a recently created plasmid, restreak some of your original miniprep culture to obtain single colonies. I always go from single colonies on a plate.

-Establish seeding cultures by picking 4 single colonies for each plasmid. Pick each colony into 3ml LB+amp. Allow to grow between 5-8 hours during the day. (The extra colonies will insure a good grower for seeding--should one of them prove non-amp resistant you can still proceed.)

-Before leaving for the day, seed 1 or 2 of the cultures 1:100 in LB+amp. (I normally do 250 ml large scale cultures. At 1.5-3 µg of plasmid per milliliter of culture this assures 400-500 µg of final purified plasmid.)

-Allow to grow overnight at least 12 and not more than 16 hours. Remove some for a miniprep and 0.85 ml for a glycerol stock. (I've had some plasmids that would not grow in large scale, so I prefer to check them by miniprep before I do a bunch of Qiagen preps)

-Spin down at 6000 rpm in the Beckman or Sorval. Dump sups and invert tubes over paper towel for a few minutes, then wipe out the inside of the tubes with clean kimwipes. Cultures can be stored -20 forever at this point. (I often queue up several plasmids, even keeping backups in the freezer in case something happens while preparing the DNA for transfection.)

-At this point I follow the Qiagen instructions for tip-500s using the maxi prep protocol. I prefer to use polycarbonate Oakridge tubes for the final DNA spin. (I reserve these tubes for my own exclusive use that I wash out myself--DNA is very sticky and you do not want to transfect someone else's DNA into your cells.)

-After the final spin I visualize the pellets, marking them with a pen on the tube. I then apply 1 ml of 70% ETOH and gently rinse the pellet, inverting the tube and allowing it to air dry for about 5 minutes. The pellets are large and sticky at this point so I apply 0.8 ml of regular TE to the pellets and keep them on their sides for 8 hours to overnight in the fridge to allow complete resuspension.

-Each preparation is split in half: 0.4 ml goes into two Eppendorf tubes with 16.7 µl of 5 M NaCl to yield 0.2 M final. Two milliliters of 95% ETOH is then applied to the sides of the Oak ridge tube. After rinsing the Oak ridge tube several times with the ETOH, I then put 1 ml per Eppendorf tube, mix (at which points a precipitate should be observable), and store the Eppendorfs in the -20 for 20 min-1hour. Spin down and wash as usual, allowing to air dry for 5 min. Put each pellet in 100 µl of regular TE and store 8 hours-overnight in the fridge to allow complete resuspension. DNA can be stored at -20 forever at this point.

-Having milligram quantities of supercoiled DNA in the -20 is my minimum criteria for thawing ES cells and preparing them for transfection.

DNA for gene targeting and stable transfection needs to linear--cut and purify using the following protocol. DNA for transient transfection is typically circular, and should be further purified using the second protocol below.

Cutting And Purifying DNA For Transfection/Targeting

-Normally targeting vectors need to be linearized before transfection. I prefer to linearize fresh before a transfection, and as the cells grow for 5 days there is plenty of time to do this (and even do it twice if need be).

-To each 1/2 of a prep, containing approximately 200-250 µg of supercoiled DNA, add the following:

40 µl of 10 x Enzyme buffer

50-100 units enzyme (I prefer a new tube of enzyme for this)

bring up to 400 µl with sterile ddH2O.

Digest 3 hours-overnight (with addition of more enzyme)

Remove a small aliquot to ensure complete digestion.

-Extract the 400µl of DNA with an equal volume of 1:1 phenol:Chloroform-IsoamylAlcohol (CIA: this is 24 parts chloroform and 1 part isoamyl alcohol).

-The sup is extracted once with CIA and the final supernatant is precipitated with NaCl and ETOH as usual.

-On the final wash with 70% ETOH take the tube into the hood and remove the sup with a P1000.

-Allow to air dry 5 min and apply 104µl of Low EDTA-TE (10 mM Tris-HCl pH 8.5; 0.1 mM EDTA).

-Store tube in the fridge for at least 8 hours to resuspend.

-Remove 4 µl and run some out on a gel and do the usual A260/280 nonsense.

-I prefer clean DNA, but I don't throw out dirty stuff in case I need it in a pinch. (remember this is the ONLY part of the process you actually control.)

-Keep linearized DNA in the fridge until consumed. You will need 40 µg of linearized DNA per transfection, so it quickly gets used up.

Prepping DNA For Transfection Of Circular DNA

Supercoiled expression plasmids, like those for Cre recombinase, are prepared as follows:

-To the 100 µl of DNA add 300 µl of sterile ddH2O and extract twice with an equal volume of 1:1 phenol:Chloroform-IsoamylAlcohol (CIA: this is 24 parts chloroform and 1 part isoamyl alcohol).

-The sup is extracted once with CIA and the final supernatant is precipitated with NaCl and ETOH as usual.

-On the final wash with 70% ETOH take the tube into the hood and remove the sup with a P1000.

-Allow to air dry 5 min and apply 104 µl of Low EDTA-TE (10 mM Tris-HCl pH 8.5; 0.1 mM EDTA).

-Store tube in the fridge for at least 8 hours.

-Remove 4 µl and run some out on a gel and do the usual A260/280 nonsense.

-I prefer clean DNA, but I don't throw out dirty stuff in case I need it in a pinch. (remember this is the ONLY part of the process you actually control.)

-Aliquot your final purified expression plasmid in convenient quantities--usually 25-50 µg per aliquot.

-Store -20 in a special "electroporation ready" box.