DNA Isolation Protocols for P1 Clones
Revised 9/98 by Barret Pfeiffer. This is a rapid alkaline lysis miniprep method for isolating DNA from large PAC clones. It is a modification of a standard Qiagen-Tip method that uses no organic extractions or columns. The method works very well for doing analytical restriction digests of PAC clones and can be scaled up if necessary. With slight alterations this procedure can also be used for routine analysis of M13 RF, plasmid, and cosmid DNAs.
P1 (Filter Sterilized)
15mM Tris, pH 8.0
*****100 ug/ml Rnase A
*****The P1 solution is stable at room temperature without the Rnase added. Before doing the prep remember to add the Rnase and keep the solution on ice!!!!!!
*P2 (Filter Sterilized, room temperature)
*****Qiagen P2 good for about 2 months-better to make fresh before every prep
3M KOAc, pH 5.5
Qiagen Solutions, P1, P2, and P3, can be used!
For optimal results the protocol is divided into two (2) days
Using a sterile toothpick(pipet tip), inoculate 5 ml of LB media supplemented with Kan (50ug/ml). Grow overnight at 37 C, no longer than 18 hours. If the tubes are in a wheel set the speed at 6 for proper airation.
Place 1.5 ml of overnight in an eppendorf tube, spin at 4,000 rpm for 7 minutes at room temperature.
Discard supernatants. Resuspend (by gently pipeting up and down) each pellet in cold 0.25 ml P1 solution. Add 0.25 ml of P2 solution and gently invert the tubes 3 times. Let sit at room temperature for 20 minutes. The appearance of the suspension should change from very turpid to almost translucent.
Slowly,drop by drop, add 0.25 ml P3 solution. Plae the tubes between two eppendorf racks and give them 3 hard shakes. A thick white precipitate of protein and E.Coli DNA will form. Place the tubes on ice for 20 minutes.
Spin the tubes for 10 minutes at 10,000 rpm at 4 C. Carefully remove the supernatant, try to avoid drawing up any precipitate, and place in fresh eppendorf tubes. Add 1 ul Glycogen. Repeat step 5 once.
While the tubes are in their last spin place 0.7 ml of room temperature isopropanol in fresh eppendorf tubes. Remove the samples from the centrifuge and transfer the supernatant to the tubes containing isopropanol. Mix by slowly nverting the tubes 3 times. At this stage, samples can be left at -20 C overnight.
Spin preps in cold microfuge max speed for 30 minutes.
Pour off the supernatant and add 0.5 ml of cold 70% EtOH to each tube. Invert the tubes several times to wash the DNA pellets. Spin in cold microcentrifuge for 5 minutes. Repeat step 8 once.
Pipet off as much supernatant as possible. Occasionally, pellets will become dislodged from the tube so be careful.
Air dry pellets at room temperature in the hood for 30-60 minutes. When the pellets turn from white to translucent in appearance, i.e., when most of the ethanol has evaporated, resuspend each in 14 ul ddwater. Do not use a narrow bore pipet tip to mechanically resuspend the DNA sample; rather allow the solution to sit in the tube at room temperature, for 1 hour, with occasional tapping (at least 5 taps an hour).
Use all 14 ul of DNA/water mixture for a restriction digest.
For applications such as ligations a quarter of the resuspension will yield acceptable results.
BDGP-Modified Qiagen P1 Purification Protocol
This procedure has been adapted by Ali Moshrefi of the BDGP from the QIAGEN protocol. Please read the background information and the protocol notes in the QIAGEN plasmid Handbook before starting.
This protocol is used to isolate 100-kb P1 DNA (pAdsacBII with an 80-bp insert) from E. coli strain NS3529 (cre+). Yield of P1 DNA is typically 10-50 µg from 500 ml culture. For higher yields, transduce to E. coli strain NS3516 (cre-).
Inoculate 3 ml of 2x YT containing 25 µg/ml kanamycin with a single colony from a selective plate and grow overnight, shaking at 37°C.
Inoculate 500 ml of 2x YT containing 25 µg/ml kanamycin with 0.5 ml of the overnight culture. Shake at 37°C for approximately 3 h until an A550 of 0.15 is reached. Be sure to measure cell density and follow recommended A550 readings to obtain optimal yields.
Add 5 ml of freshly made, filter-sterilized 0.1 M IPTG to the 500-ml culture. Shake for a further 3 h at 37°C or until an A550 of 1.3-1.5 is reached. IPTG is added to induce P1 replication.
harvest cells by centrifugation at 5000 x g for 10 min. Cell pellets can be stored overnight at -20°C following complete removal of the supernatant.
Resuspend the bacterial pellet in 20 ml Buffer P1*. Be sure to add RNase A (100 µg/ml) to Buffer P1 before use.
Add 20 ml Buffer P2*, mix gently by inverting 4-6 times, and incubate at room temperature for 5 min. Check Buffer P2 before use for SDS precipitation due to low storage temperatures. If necessary, dissolve the SDS by warming to 37°C.
Add 20 ml chilled Buffer P3*, mix immediately but gently by inverting 4-6 times, and incubate on ice for 15 min.
Centrifuge at ³20,000 x g for 30 min at 4°C. Remove supernatant promptly.
Re-centrifuge at ³20,000 x g for 15 min at 4°C. Remove supernatant promptly (Alternatively the supernatant can be filtered over a prewetted, folded filter).
Equilibrate two QIAGEN-tip 500's by applying 10 ml Buffer QBT, and allowing the column to empty by gravity flow.
Split the supernatant from step 9 into two equal parts and apply to two QIAGEN-tip 500s and allow supernatant to enter the resin by gravity flow.
Wash each QIAGEN-tip column with 2 x 30 ml Buffer QC.
Elute DNA with 15 ml Buffer QF over each column. (Prewarming Buffer QF to 50°C before elution may increase yield, but we have never tried this).
Precipitate by adding 10.5 ml room-temperature isopropanol to each aliquot of eluted DNA and mixing gently. Centrifuge immediately at >15,000 x g for 30 min at 4°C, and carefully remove the supernatant.
Wash the DNA pellet 2 times with 5 ml ice-cold 70% ethanol and centrifuge at >15,000 x g for 10 min each time.
Carefully remove the supernatant and air-dry for 5-10 minso no or very little liquid is left in tubes. Caution: Do not overdry DNA pellet after washing with 70% ethanol.
Redissolve each pellet in 300-500µl TE, pH 8.0 or ddH2O. Cut off the bottom 0.5 cm of a 1-ml pipette tip to resuspend. This step avoids shearing the high molecular weight DNA. DNA resuspends instantly, but it is fine to let sit for 30 min at room temperature with occasional gentle tapping.