Protocols for the CEQ 2000

For diagram of Library process click here: diagram

Large Scale BAC Protocol

1. Inoculate 25 ml culture of TB containing 10ug/ml Chlorophenicol with a PCR positive BAC colony. Incubate overnight in 37° shaker.

2. Inoculate 2-500 ml culture og TB containing 10ug/ml Chlorphenicol with a 12 ml of the BAC culture. Incubate overnight in 37° shaker.

3. Centrifuge 500 ml of an overnight culture in 250 ml bottles at 5000 rpm for 10 min at 4°C. (Make Glycerol Stock if necessary)

4. Resuspend in 20ml fresh lysozyme solution (50mM Glucose, 25mM Tris pH8, 10mM EDTA plus 5mg/ml lysozyme), place tube on ice for 10-20 min.

5. Add 40ml fresh alkaline SDS (1% SDS, 0.2 N NaOH), mix gently and keep pn ice for 5 min.

6. Add 30ml Potassium Acetate (pH 4.8), mix well and keep on ice for 15-30 min.

7. Centrifuge at 10,000 rpm for 40 min at 0°, collect supernatant in a clean 250 ml bottle.

8. Add 2 volumes 100% EtOH and mix well.

9. Centrifuge at 10,000 rpm for 20 min at 4°C.

10. Pour off supernatant, rinse pellet with cold 70% EtOH, drain bottles, and dry in dessicator for 15-20 min.

11. Resuspend pellet in about 4ml TE for a 1L culture.

12. Add 0.1 volumes of 8M LiCl mix and place 2 ml in each tube for TLS-55 rotor. Spin at 20,000 rpm for 30 min at 2°C.

13. Remove supernatant and divide between 4-1.5 ml eppindorf tubes. Add 2 volumes EtOH and spin in eppindorf centrifuge for 15 min. Wash with 70% and dry in speed-vac. Resuspend pellets in 50ul TE.

14. O.D. sample and do a restriction digest.

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Large Scale PAC Protocol

Day 1:

Add a single colony into 50 ml/ 250 ml flask. Seed LB containing 25 ug/ml kanamycin with bacteria harboring PAC. Grow overnight to stationary phase, shaking 300rpm at 37°C.

Day2:

To 500 ml TB+Kan / 2000ml flask, add 16.6 ml overnight culture. Shake 1.5 hours. Add IPTG (600ul/500ml growth) - IPTG stock solution=200mg/ml, continue to grow for 5 hours, then harvest. Centrifuge in 250 mL bottles, at 10,000xg for 5 min. Discard supernatant.

Flash freeze pellet in liquid N2, store in -80°C.

1. Resuspend in 20 mL GTE (50mM glucose, 25mM Tris pH8, 10mM EDTA). Add 600uL of 50mg/ml lysozyme in TE suspension. Mix gently.

2. Incubate at room temp for 5 min.

3. Add 40 ml freshly made 0.2M NaOH/1% SDS, swirl with pipet then place at room temp for 5 min.

4. Add 30 ml of 3M KOAc. Swirl with pipet then place on ice for another 30 min. Mix every 10 min.

5. Centrifuge at 13,000 rpm for 25 min at 0°C. and collect supernatant in a clean 250 ml bottle. Centrifuge second time if necessary.

6. Add 0.6 volumes isopropanol and let sit on ice for 20 min.

7. Centrifuge at 14,000 rpm for 25 min.

8. Pour off supernatant, rinse pellet with ice cold 70% EtOH, drain tubes and dry in a dessicator for 15-20 min or until dry.

9. Resuspend pellet in about 10ml TE with CsCl (R.I. 1.39). Best results are achieves when resuspending overnight at 4°C.

10. Transfer to a 14ml falcon tube and add 150uL of ewthidium bromide (10mg/ml). Spin at 1700xg in a tabletop centrifuge for 5 min to pellet debris. Transfer supernatant to a polyallomer quick-seal tube and fill to neck with CsCl in TE (R.I. 1.39) and seal.

11. Centrifuge in Ti90 rotor in ultrcentrifuge at 70,000 rpm, 20°C, deceleration at 9 or slow for 20 hours.

12. Remove lower, plasmid DNA band, and mix with saturated isopropanol to remove EtBr, discard top layer and repeat until sample is clear. (It is wise to pull top band also and clean)

13. Dialyze against 1000 volumes of TE pH8 overnight changing buffer at least once.

14. Precipitate DNA with EtOH/NaOAc and resuspend in TE pH8. Take O.D.

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Insert Protocol/Prep

1. Shear genomic DNA using Nebulizer.

Dilute sample to 25 ug/ml in 2mls of TE. Run DNA through Nebulizer at 150 psi nitrogen for 2.5 minutes. Remove sample then add 2 mls TE to device and run briefly to rinse. Collect wash and pool with the original sample. Run an aliquot on an agarose gel to assess shearing effiency. Speed-vac down to ~0.5ml or less.

2. Mend ends of sheared genomic DNA.

Set up a 200uL rxn with 25ug DNA, 0.2 mM dNTP's, and 40 units T4 DNA Polymerase. Incubate at Rm. T. for 15 min. Add 25 units Klenow and incubate at Rm. T. for 1 hour longer. Stop reactions by incubating sample at 72°C. for 10 min. Cool to Rm. T. then add 20 uL 3M NaOAc to the mended pathogen DNA. Extract with phenol/chloroform once each. Add EtOH and precipitate. Resuspend rinsed and dried pellet in 20uL TE.

5X mending buffer

335mM Tris pH 8.8

83mM (NH4)2SO4

33.5mM MgCl2

33.5 EDTA

835 ug/ml BSA

50mM ß-mercaptoethanol

3. Size Fractionate fragments

Establish the preferred range of insert sizes for the library then prepare an appropriate agarose gel. Suggested isolation: 300-800 bp fragments using a 1.5% gel. Load sample onto agarose gel. Fractionate and cut out the desired inserts. Electroelute DNA and collect dialysate. Remove a ample for a fluorimeter quantitation, then phenol/chloroform extract. Precipitate sample.

4. Phosphorylate and purify inserts for ligation into vector.

To phosphorylate the random fragments, resuspend sample in dH20 and set up a 100uL reaction in T4 DNA kinase buffer with 2mM ATP, and 20 units T4 polynucleotide kinase. Incubate at 37°C for 30 min. Heat inactivate the kinase at 72°C for 15 min.

10x T4 polynucleotide kinase buffer:

700mM Tris-HCl, pH 7.6

100mM MgCl2

50mM DTT

To purify insert for ligation, exchange the reaction buffer and remove any excess ATP by passing the sample through a QuiQuick column (Qiagen, Inc.). The samples can also by purified by passing through a 1ml G-50 Sephadex column. Add 0.3 M NaOAc and precipitate in ethanol.. Spin and resuspend in dH2O. Measure concentration.

7. Set up test ligations.

The optimal insert to vecto ratio will depend on your vector and insert sizes and the efficiency of your vector and insert preparations. As a rule of thumb, a 5:1 or 10:1 molar ratio of insert to vector with at least 10ng/uL of both in a reaction works well.

8. Choose the desired ligation ratios, scale-up, and ligate.

*Be sure to transform into a RecA- strain to prevent any problems later.

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Transformations

GibcoBRL Life Technologies transformations kits are easy and convenient to use.

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Testing Bacteria for alpha-complementation

1. To premade LB plate containing appropriate antibiotics, add 40 uL of a stock solution of X-gal (20mg/ml in dimethylformamide) and 4uL of a stock solution og IPTG (200mg/ml). Higher concentrations of both can be added to LB before the plate in poured.

2. Using glass spreader, spread solution over the netire plate.

3.Inoculate the blate with the bacteria. Streak with 100uL of a suspension of bacteria.

4. Incubate overnight at 37° C and then place at 4° for several hours to develop color.

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HYT Media (for glycerol stocks of colonies)

Recipe for 10 L:

1. In 9L dH2O add:

160 g Bacto-tryptone

100 g Yeast extract

50 g NaCl

2. Adjust pH to 7.0 and then add:

62.7 g K2PO4 (36mM)

17.9 g KH2PO4 (913mM)

5 g Sodium Citrate . 2H2O

4 ml 1M MgSO4 (0.4mM)

9 g (NH4)SO4 (6.8mM)

352 ml glycerol (4.4% w/v)

3. Fill to 10L w/dH2O

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96-well Plate EtOH Precipitation

1. ADD 4uL of stop solution (1.5 M NaOAc + 50mM EDTA) to your cycle sequencing rxn and 1uL of 20ug/uL glycogen.

2. ADD 60uL cold 95% EtOH

3. Spin at 3200rpm for 1.5 hours.

4. Remove supernatant

5. ADD 200uL 70% EtOH