Phage DNA preparation

 

To 750 uL of high-titer phage suspension, 1 uL of DNAse [Fisher] is added.  2 uL of 50 mg/mL RNAse[Boehringer...] is also added.  This solution is then incubated at 37C for 10 minutes.  150 uL of STEP Lysis buffer is added.  10 uL of 10mg/mL proteinase K (PK)[Boehringer...] is then added. 

The sample is then incubated for 30 minutes at 65C, this step inhibits the DNAse, RNAse and PK enzymes. 

750 uL of Chloroform:IsoAmyl Alchohol:Phenol (24:1:24) [ready-made from...] is added, shaken well and the solution is then centrifuged at 10,000 rpm for 5 minutes.  The upper layer is then removed by careful pipetting with a micro-pipette, and  750 uL of chloroform:isoamyl alchohol (24:1) [made fresh] is added to this upper layer, shaken, and centrifuged for 5 min at 10,000 rpm. 

The upper layer is then removed and placed in a new centrifuge tube (about 600 uL).  1/10 th of the volume of the upper layer is given 0.3 M Sodium Acetate (a 3 M Sodium Acetate solution, pH 5.1 is the stock, and .7 volumes of 100% isopropanol.  The solution is then allowed to sit for at least 2 hours (in a freezer is best, a fridge is ok, and room temperature will probably work), although longer periods such as overnight are acceptable.  This is a great place to stop.  See Note about when you can take an intermission during this procedure.  The tube is then centrifuged for 5 min at 10,000 rpm and the supernatant is removed by pipetting and if possible, under vacuum. 

The pellet is then washed in 1mL 70% ethanol then centrifuged for 5 min at 10,000rpm. The ethanol is then removed and the pellet is then placed in a rotovap on low to remove any existing ethanol. The pellet is then suspended in 100 uL (or 10 uL's if higher concentration of DNA is needed) of deionized water and dissolved at 55C, this can take more than an hour.  DNA can be stored in the fridge at 4C. 

The above is done in microcentrifuge tubes that have been autoclaved.

Solutions and Solution prep notes:

STEP Lysis buffer:

EDTA, 0.5 M            400 uL  [Fisher biologics is a very good supplier of chemical reagents.]

10% SDS, 10%           50 uL

Tris (pH 8.0), 1M      25 uL

Distilled water         25 uL

IMPORTANT: heat the EDTA to 55C before adding the other substances, otherwise the SDS precipitates.  Some precipitation can be rectified by placing the Lysis buffer at 55C for a couple minutes. 

NOTES:

After centrifugation, and formation of upper and lower liquid partitions, an inter-phase will form.  This white layer should not be removed with the DNA that is found in the upper aqueous layer. 

It is important to get this step going quickly, as about 3 3/4 hours of uninterrupted lab-work is needed, or should be planned on, prior to getting DNA into isopropanol.  The procedure cannot be stopped at an earlier step.  The remainder of the procedure can be done the next day. 

Drying DNA can take quite a while, at AECOM we use a rotovap to do this.  This is a centrifuge under vacuum, with a temperature gradient between the centrifuge and another compartment.  By having a temperature gradient between the sample (heated) and collecting reservoir (the other compartment), the rate of evaporation of any liquid is increased.  One note: even though it is okay to heat DNA in order to vaporize liquid away, I don't like to cook my DNA too much.  I believe that too much heat, for too long, will damage the DNA.  Having pipetted as much of the 70% ethanol away from my DNA sample, 10 uL or so of the liquid still remains.  With the rotovap pre-heated at the highest setting, he compartment is (seemingly at 65C or so) a little warm for my taste.  Our DNA samples looked pretty good after rotovapping for 5 minutes after preheating and leaving the heating element on the highest setting, followed by turning the heating element off and rotovapping for an additional 5 minutes.  I stopped the rotovap after the first 5 minutes to look at the status of evaporation.  After the 10 minutes, the samples were very dry, and nice DNA bands were visible on a 0.8% agarose gel. 

**We recommend that before analyzing and comparing DNA fragments that have been digested with restrction enzymes, that the concentration and existance of the phage DNA be verified rapidly electrophoresing in an agarose gel (see this restriction digestion link for electrophoresis recommendations). Although this is an opportunity to teach spectrophotometric techniques, sensitive equipment will be needed to measure the small quantities of DNA harvested from the phage. The gel voltage can be turned up significantly, and the presence and gross quantity of the DNA can be rapidly visualized. This procedure makes sense when considering the lab, cost and time of performing restriction digestions.