Preparation of BAC DNA with by Alkaline Prep Method

CLONE ISOLATION PREP - PROTOCOL

Before you begin isolating DNA from the cultures:

--GENTLENESS is key through step 10!

--Try to do prep steps 1-8 at the side of the centrifuge

--Make Solution III and place in fridge or on ice morning of isolation

1. Grow 250 ml of 2X YT culture at 37 ℃for 16 hours (> 250 rpm) with the appropriate antibiotic. Cultures can be started by 1) picking a single colony or 2) by splitting a subculture into 250 ml of culture (1 L Flasks). Colonies should be picked within few days.

*****Other prep sizes may change yields and purity*****

2. Transfer the culture to a 250 ml centrifuge bottle (polypropylene, washed and autoclaved; lids scrubbed with Alconox) and spin at 6000 g's for 15 minutes at 4℃(fixed angle; e.g. JA-14>6.2K RPM). Before this spin, make a glycerol stock from the culture. After the spin is finished, dump off the supernatant, drain the bottle briefly upside down on paper towel, and place the bottles containing the pellets on ice before proceeding to the next step.

3. Push all of the pellet off the side of the tube using the end of a 10 ml pipette, leave the pipette in the bottle. Do this for all the samples. Add 20 ml of Solution I (10 mM EDTA, pH 8.0; Room temperature) to every sample and pipette until the pellet is re-suspended.

4. After all the pellets have been re-suspended (go back to 1st bottle, re-pipette 1-2x to look for clumps), move the bottles from the ice to the bench top. Let them sit for 5 minutes.

Solution II: 0.2 M NaOH, 1% SDS (250 ml-> 5 ml 10 M NaOH, 25 ml 10% SDS, and 220 ml water)

5. Add 40 ml of Solution II (prepared fresh) allowing it to run down the sides of the bottle using 360° motion around top of bottle over about 5s. Hold bottle firmly in place during this addition.

DO NOT VORTEX OR MIX AT THIS STEP!!!! Allow the samples to sit at room temperature for 5 minutes. Be sure to start your timer when you have finished adding Solution II to the first sample. DO NOT LET THE SAMPLES SIT FOR MORE THAN 5 MINUTES BEFORE PROCEEDING TO THE NEXT STEP!!!!!!!!!!

Solution III: 200 ml-> 50 ml 7.5 M K-acetate, 23 ml Acetic Acid, and 127 ml water. Make at start of day and put in fridge or on ice so is cold when get to this point.

6. Add 30 ml of Solution III allowing it to run down the sides of the bottle using 360° motion around top of bottle over about 5s. Hold bottle firmly in place during this addition. Gently move the bottle from the bench top to the ice. Set a timer for 15 minutes and start it before moving on to the next sample. DO NOT VORTEX OR MIX AT THIS STEP!!!!!!! DO NOT LET THE SAMPLES SIT FOR MORE THAN 15 MINUTES BEFORE PROCEEDING TO THE NEXT STEP!!!!!!!!!!

Should see white precipitate and white streaks in upper phase. Any brown or the absence of streaks are bad signs and you probably should discard that prep at that point.

7. Spin at 30,000 g's (fixed angle; e.g. JA-14>14.0K RPM) for 20 minutes at 4 °C, remove supernatant into a clean bottle, avoiding the protein. Repeat the spin and again remove the supernatant into another clean bottle. The supernatant should be clear with no white clumps. The transfers are done by pouring with pellet on upper surface.

8. Add 45 ml isopropanol (less than one month since opened) to the supernatant allowing it to run down the sides of the bottle using 360° motion around top of bottle over about 5s. Hold bottle firmly in place during this addition. Spin at 6500 g's (fixed angle; e.g. JA-14>6.5K RPM) for 15 minutes, placing a landmark on the bottle to the outside so you will know where to find the pellet.

9. Pour off the supernatant and place bottles on paper towel to drain briefly. Add 9 ml of 10 mM Tris/50 mM EDTA down side with pellet to dissolve the pellet. Swirl the pellet gently for a few minutes. If this doesn't work, the bottles may be heated at 37℃in an oven for 10 minutes and then use horizontal rocking to dissolve pellet. Alternatively, the 10T/50E solution could have been pre-warmed to 37℃and used to dissolve the pellet.

DO NOT VORTEX AT THIS STEP!!!!!!!!

Transfer the DNA solution to a 50ml capped tube by pouring, making sure all of it is transferred. Add 4.5 ml of 7.5 M K-acetate, mix gently horizontally until phases mix, and place at -70 ℃for 30 minutes.

10. Thaw at room temperature and spin at 4000 g's in swinging bucket for for 10 minutes. While this is spinning add 27 ml of 100% ethanol to another set of 50ml capped tubes. When the spin is finished, pour the supernatant over the 27 ml of ethanol and mix gently horizontally until phases mix.

11. Spin at 4000 g's in swinging bucket for 10 minutes. Pour off supernatant and drain or wipe out excess supernatant. Should see pellet at this point. Dissolve the pellet in 700 µl of 50T/50E (50 mM Tris/50 mM EDTA, pH 8). Add 10 µl of RNase (10mg/ml) and leave at 37℃in a water bath for 1 hour.

12. Transfer the DNA solution to a 1.5ml microcentrifuge tube and add 700 µl of phenol:chloroform, vortex gently until the phases mix, spin for 5 minutes (microfuge top speed, room temperature) and pipette the top layer into a clean microcentrifuge tube. Be sure to not transfer the white interphase.

13. Extract once more with 700 µl of phenol:chloroform: vortex gently until the phases mix, spin for 5 minutes and pipette the top layer into a clean microcentrifuge tube. Be sure to not transfer the white interphase.

14. Add 700 µl of chloroform, vortex gently until the phases mix, spin for 5 minutes and pipette the top layer into a clean microfuge tube, being sure not to transfer any white interphase.

15. Add 700 µl of isopropanol, vortex gently until mixed and spin for 20 minutes. Discard the isopropanol, being sure not to lose the pellet. Wash the pellet with 500 µl of 70% ethanol, remove excess by pipeting and air-dry for 30 minutes.

16. Dissolve the pellets in 100 µl of TE, pH 8.0.

17. Quantitate on the flourometer (use pico green) or spectromphotometer. Should get 4-7µg per 250ml culture that is 95-98% BAC DNA.

18. Check for E. coli, by loading 2 ml on a 0.8% agarose gel in TAE, run > 63 V for 1 hour.

Reagent List

2X YT (Quality Biologicals, 340-023-100)

Glycerol stock: 400µl 50% glycerol and 400µl culture

10mM EDTA, pH 8.0

10% SDS

5N NaOH

** 7.5M potassium acetate (Sigma, P-3542)

Glacial acetic acid

RNase A (heat inactivated) (LTI or Sigma)

Isopropanol

10mM Tris/50mM EDTA, pH 8.0

50mM Tris/50mM EDTA, pH 8.0

** Phenol:chloroform:IAA(25:24:1) (Sigma, P-2069)

Chloroform

** indicates reagent should be from a specific company (not tested for relevance by us)