DNA preps

Large gram-negative DNA prep (phenol version)
Gram-negative DNA mini prep (salt version)
Gram negative ammonium acetate mini plasmid prep
Boil prep for PCR
Column prep for isolation of DNA from agarose gel slices
CTAB prep for plant genomic DNA

Large gram-negative DNA prep (phenol version)

Use cut tips when transferring genomic DNA to new tubes

1. Grow a 3 ml culture overnight at required temp in appropriate medium.
2. Inoculate 50 ml with 50 ul of overnight culture and grow to stationary phase.
3. Place phenol/chloroform at 4°C.
4. Transfer culture to two 50 ml Blue max, Sarstadt or Oak Ridge tubes
5. Pellet cells by spinning tubes at 3000 x g for 10 minutes.
6. Discard the supernatant and drain the tubes upside down on a paper towel.
7. Resuspend the bacterial pellet in 10 ml of T50E50.
8. Add 3 ul Ready-lyse (Epicentre) and 10 ul of Rnase A (Sigma R4642).
9. Incubate at room temp for 15 min, then on ice for 15 min.
10. Add SDS to 1% final concentration and Pro K to 1 mg/ml final concentration (this is equivalent to 500 ul of 20% SDS and 100 ul of a 100 mg/ml Pro K solution)
11. Incubate at 50 or 37 C for 1 hour.
12. Add ammonium acetate to a final concentration of 2.5 M
13. Add Phase Lock Gel to approximately 5 ml
14. Add equal volume of phenol/chloroform/IAA.
15. Mix by gentle inversion for 10min.
16. Centrifuge 10 min 8000 x g (If necessary, repeat phenol chloroform extraction).
17. Pour supernatant into new tube and re-extract with chloroform/IAA.
18. Add 2 volumes of room temp 100% ethanol and gently rotate the tube until solutions are completely mixed. The DNA should precipitate as long strands. Transfer the DNA to a 15 ml tube containing 3-5 ml of 70% ethanol.
19. Allow to sit at room temperature for 15-30 min.
20. Pour off the ethanol and remove the excess with sterile swab. Resuspend the DNA in a moderate amount of TE
21. Add one drop of chloroform and store at 4°C;. Cleaner DNA can be obtained with dialysis (see below)
22. Run a 1 x TAE .4% gel with bromphenol blue as marker
* Do not allow DNA to completely dry as re-suspension will be impossible. A moderate amount is ˜ .250-1 ml.

DNA dissolving tricks-Incubate tube at 55°C 10´ then put at 37°C or room temp for 1 hour to overnight. If not dissolved try 55C° for 10-20 min or add more TE.

Optional Dialysis

1 Pour solution into 25,000-50,000 MWCO dialysis tubing. **
2 Dialyze against T10 E10 (10mM pH 8.0 Tris 10mM EDTA) for 12 hours with 2 changes and 10mM Tris 2mM EDTA for 12-48 hours (I use about 1- 2 liters per 1-3 dialysis tubes.)
3 To reduce volume sprinkle dry sepharose G150-200 on the tubing until the desired volume is reached. (˜500-750. µl)***
4 Add one drop of chloroform and store at 4°C°.

**I used Spectrapor Dispodialyzers MWCO 25,000 1 ml part # 50,000

*** I use G150 or whatever I can get from chemical surplus. It forms a skin which should be peeled off every so often and pitched

(last update; Andy Witherell, Oct 2004)

Gram-negative DNA mini prep (salt version)

1. Spin down 2-4 ml of culture in a 2 ml centrifuge tube at 5000 x g for 5 min.
2. Resuspend pellet in 1 ml of T50E50 (pH 8.0) buffer and .5 ul of Ready-lyse (Epicentre) and 2.5 ul of Rnase A (Sigma R4642).
3. Incubate for 15 min at room temp, then for 15 min on ice.
4. Add SDS to 1% and Pro K to 1 mg/ml (this is equivalent to 50 ul of 20% SDS and 10 ul of 100 mg/ml Pro K).
5. Incubate at 55 C for 1 hour.
6. Add 270 ul of saturated NaCl and rotate tubes for 2 min.
7. Centrifuge tubes at 5000 x g for 10 min.
8. Transfer the supernatant to 5 ml tubes.
9. Add 2 volumes of room temp 100% EtOH and gently mix phases. The DNA should precipitate in long strands.
10. Remove supernatant and wash DNA once with 70% EtOH, then twice with 100% EtOH.
11. Remove supernatant, swab off excess, dry slightly and resuspend the DNA in a moderate amount of TE.
12. For cleaner preps reppt with 2.5 M NH4AC and 2 volumes of 100% EtOH.

When loading gels, if excess EtOH is in the DNA, add loading dye, place on parafilm and move around with tip to absorb excess.

T50E50 10ml
500 ul 1 M Tris (pH 8.0)
1 ml 0.5 M EDTA
8.5 ml H2O

(last update; Andy Witherell, Oct 2004)

Gram negative ammonium acetate mini plasmid prep

1. In a 2 ml centrifuge tube, spin down 4 ml of an overnight culture for 1 min at max speed.
2. If using for sequencing, respin culture and remove residue supernatant.
3. Add 200 ul solution I to pellet and resuspend using vortexer. Incubate at room temp for 5 min.
4. Add 400 ul solution II and invert. Incubate on ice for 5 min minimum (maximum 10 min).
5. Add 300 ul ice cold 7.5 M NH4AC pH 7.5 and invert. Incubate on ice for 5 min
6. Spin tubes for 5 min at room temp.
7. Pour supernatant into new tubes.
8. Treat with 2 ul RNase at 37 C for 20min (Sigma R4642).
9. Add 10 ul of a 100mg/ml stock of proK and incubate at 37 C for 20 min.
10. Add 270 ul of saturated NaCl.
11. Rotate tubes for 2 min or shake tubes for 5-10 seconds.
12. Spin tubes at 3000 x g for 10 min.
13. Transfer the supernatant to a 2 ml tube.
14. Add 0.6 x isopropanol (700 ul) to each tube and incubate at room temp for 10 min.
15. Centrifuge tubes at high speed for 10 min at room temp.
16. Wash with 70% EtOH.
17. Take off supernatant, swab off excess, and resuspend in 25-50 ul of TE.
18. For cleaner preps, re-precipitate with 2.5 M NH4AC and 2 volumes EtOH.

Solution I
For 6 ml (30 mini preps)
16.8 ml water
50 ul 1M Tris pH 8.0
40 ul 0.5 M EDTA
50 ul 2 M glucose
0.5 ul/ml Ready-lyse (Epicentre)

Solution II
For 24 ml (30 mini preps)
22.4 ml water
120 ul 10 N NaOH
300 ul 20% SDS

7.5 M NH4Ac 100mls pH 7.6
To 50 ml of dH20 add 57.8 g of NH4Ac. pH to 7.6 with NH4OH or glacial acetic acid. Bring to 100 ml. Test the pH again. Filter sterilize. Store at 4 C°, tightly capped.

(last update; Andy Witherell, Oct 2004)

Boil prep for PCR

1. Fill epi-tube with 500 ul of sterile water.
2. Scrape a few colonies from a fresh plate of bacteria and transfer the cells to the epi-tube.
3. Boil the cells for 5 min.
4. Centrifuge the tube for 5 min. at high speed to pellet the debris.
5. Transfer the supernatant to a new epi-tube and use 1 ul per 50 ul PCR.

(last update; Amy Charkowski, May 2005)

Column prep for isolation of DNA from agarose gel slices

1. Run DNA in agarose gel. Stain in new ethidium bromide solution. Visualize DNA bands on UV transilluminator and cut out target band.
2. Put gel slice into microspin filter columns 0.45 um CA/G (Alltech #24133) and spin column at top speed for 5 min. The agarose should stay on in the column and the DNA will be in the liquid flow through in the catch tube.
3. Transfer the flow through to a new 1.7 ml tube. Add 0.1 vol of 10 M ammonium acetate and 3 vol of isopropanol. Incubate for 10 min at room temp (or better, at -20C).
4. Centrifuge the tube for 5 min. at high speed to pellet the DNA.
5. Wash the DNA once with 500 ul of 70% ethanol. Resuspend the pellet in an appropriate volume of water or buffer. Usually, resuspending the DNA in TE to 90% of the original volume of the DNA solution loaded onto the gel is appropriate for ligations.

(last update; Amy Charkowski, June 2006)

CTAB prep for plant genomic DNA

1. Grind 0.5 to 1 g of plant tissue in N2 in mortar and pestle. Transfer to15 ml poly propylene centrifuge tube with an RCF of at least 6000 (9000 is preferable). Use the tube to pick up the leaf powder. Add 4.75 ml of extraction buffer heated to 60C and 1 to 5% vol. of ß-mercaptoethanol.

2. Incubate sample at 60C for 30 to 60 minutes with occasional swirling.

3. Add 1 volume Chloroform/IAA (24:1 Seavags mixture). Place tube on rotator for 5-10 minutes with gentle rotation. Allow tubes to sit upright for 10 min. When adding Seavag leave the tops off to get rid of gases.

4. Spin 1500 x g for 10 min at room temperature. (A swinging bucket rotor is preferable). The Seavag wash can be repeated once more if necessary.

5. Transfer super to a clean tube using a cut 5ml pipet tip. Add 1/5 volume 5% CTAB .7M NaCl and 2 volumes isopropanol. Incubate at RT for 1 hour.

6. Spin at 500 x g for 2 minutes. Gently pour off supernatant as much as possible. Sometimes spinning is unnecessary and you can just pour off the supernatant.

7. Wash the pellet with buffer 1 by letting it sit for 20 minutes at room temperature. Repeat this wash once.

8. Resuspend pellet in 1 ml of wash buffer and let sit at room temperature for 15 to 20 minutes. (This is a stopping step and can let the DNA go O/N.

9. Centrifuge sample at max speed for 10 minutes.

10. Dissolve in 0.5 to 2 ml lo TE. There are a number of tricks to get the DNA to go into solution: sequentially add TE, heat at 55C for 10 to 30 min and then put at 37C for 1 to 24 hours. Or allow sample to sit at 4C for a few days. Do not allow DNA to completely dry as re-suspension will be impossible. Add 1 drop of chloroform and store sample at 4C.

Notes: Cleanup steps involve adding Rnase, incubating at 37C for 30 min and ETOH/NH4Ac ppt. etc. or add equal volume of 20% PEG/1.5M NaCl incubate at 1hr, spin at max speed wash 1-2x with 70% EtOH spin RT take off supernatant slightly air dry and resuspend in TE. This step cleans it up for cutting. If the DNA is visible after ppt then just pouring off and adding of solutions w/o centrifuging can be done. I use two tubes and pour the DNA back and forth to get rid of the solutions.

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