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仿造与测序实验

文章作者:生命科学 上传时间:2019-08-27

核心提示: A. Sonication The generation of DNA fragments by sonication is performed by placing a microcentrifuge tube

核心提示:Introduction This manual is a compilation of many of the everyday methods used in the average molecular biology laborIntroduction

核心提示:Reagents:ChIP sonication Buffer :10Reagents:ChIP sonication Buffer :10 ml 10% Triton X-1001 ml 10% Deoxycholate5 ml 1 M Tris-Cl pH 8.11 ml 0.5 M EDTA3 ml 5 M NaCl80 ml WaterJust before use, add 10 ul Aprotinin, 10 ul Leupeptin, and 5 ul PMSF to each 10 ml.High Salt Wash Buffer 10 ml 10% Triton X-1001 ml 10% Deoxycholate5 ml 1M Tris-8.11 ml 0.5M EDTA10 ml 5M NaCl73 ml Water

A. Sonication

The generation of DNA fragments by sonication is performed by placing a microcentrifuge tube containing the buffered DNA sample into an ice-water bath in a cup-horn sonicator and sonicating for a varying number of 10 second bursts using maximum output and continuous power , essentially as described by Bankier and Barrell . During sonication, temperature increases result in uneven fragment distribution patterns, and for that reason, the temperature of the bath is monitored carefully during sonication, and fresh ice-water is added when necessary. The exact conditions for sonication are determined for a given DNA sample before a preparative sonication is performed. Approximately 100 ug of DNA sample, in 350 ul of buffer, is distributed into ten aliquots of 35 ul, five of which are subjected to sonication for increasing numbers of 10 second bursts. Aliquots from each time point are electrophoresed on an agarose gel versus the phi-X 174 size marker to determine the approximate DNA fragment size range for each sonication time point. Once optimal sonication conditions are determined, the remaining five DNA aliquots are sonicated according to those pre-determined conditions. After sonication, the five tubes are placed in an ice-water bath until fragment end-repair and size selection, discussed below.

Protocol

  1. Prepare the following DNA dilution, and aliquot 35 ul into ten 1.5 ml microcentrifuge tubes:

    DNA 100 ug 10X TM buffer 35 ul sterile ddH2O q.s. Final Volume 350 ul

  2. To determine the optimal sonication conditions, sonicate the DNA samples in five of the tubes in a Heat Systems Ultrasonics W-375 cup horn sonicator set on 'HOLD', 'CONTINUOUS', and maximum 'OUTPUT CONTROL' = 10 under the following conditions:

    TubeNo. 10 second bursts1122334455

We have recently learned that the Genome Center at Washington University and the Sanger Center set the OUTPUT CONTROL to the lowest possible settings. Because at present we use the Nebulizer , we have not investigated this further.

  1. Cool the DNA samples by placing the tubes in an ice-water bath for at least 1 minute between each 10 second burst. Replace the ice-water bath in the cup horn sonicator between each sample.

  2. Centrifuge the samples to reclaim condensation and electrophorese a 10 ul aliquot from each sonicated DNA sample on a agarose gel versus the phi-X 174/HaeIII size marker .

  3. Based on the fragment size ranges detected from agarose gel electrophoresis, sonicate the remaining 5 tubes according to the optimal conditions and then place the tubes in a ice-water bath.

This manual is a compilation of many of the everyday methods used in the average molecular biology laboratory, with emphasis on the techniques for large scale DNA sequencing protocols and DNA sequencing automation techniques. The manual has been written in a protocol format, with little theoretical discussion. For theory and additional information, users of this manual are referred back to the original literature, or to other textual manuals such as those published by Maniatis et al. and Glover .

25 ml 1M LiCl5 ml 10% IGEPAL5 ml 10% Deoxycholate1 ml 1M Tris-8.1200 ul 0.5M EDTA64 ml WaterProtease inhibitors Leupeptin 2 mg/ml in waterAprotinin 2 mg/ml in waterPMSF 0.2 M

B. Nebulization

You can purchase Nebulizer, Number 4101 or 4101UO, from a local supplier, whose name you can obtain by calling the manufacturer:

IPI Medical Products Inc.3217 North KilpatrickChicago, IL 60641phone:  777-0900

The president of IPI is Walter Levine so if you have any troubles ordering them be sure to ask for him and/or to tell them that these devices are: "NOT INTENDED FOR PATIENT USE"

Basically we follow a protocol sent to us by Steve Surzycki at the Department of Biology, Indiana University.

There are two small problems that we solved as follows:

  1. You have to cover the hole where normally the mouth piece gets attached to; cover that hole with a cap QS-T from ISOLAB Inc. .

  2. The other problem that may occur is that the nebulizer leaks where the hose for the nitrogen gets attached. It seems that Nalgene tubing seals better that the tubing which comes with the nebulizer. The nebulizer might still leak somewhat at the top, you can't avoid that.

Nebulizer Summary:

A nebulizer containing 2 ml of a buffered DNA solution containing 25-50% glycerol is placed in an ice-water bath and subjected to nitrogen gas at a pressure of 8-10 psi for 2.5 minutes for nebulizing BACs . Nitrogen gas pressure is the primary determinant of DNA fragment size, and although pressure studies should be performed with each BAC, cosmid or plasmid, a pressure of 8-10 psi almost always resulted in the desired fragment size range. As discussed above for sonication, the use of an ice-water bath for nebulization also is critical to the generation of evenly distributed DNA fragments. During the nebulization process, unavoidable leaks are minimized by securely tightening the lid for nebulizer chamber and sealing the larger hole in the

top piece with a plastic cap. To prepare for fragment end-repair, the nebulized DNA typically is divided into four tubes and concentrated by ethanol precipitation.

Protocol

  1. Modify a nebulizer by removing the plastic cylinder drip ring, cutting off the outer rim of the cylinder, inverting it and placing it back into the nebulizer. Seal the large hole inthe top cover with a plastic stopper and connect a 1/4 inch id length of Tygon tubing to the smaller hole.

  2. Prepare the following DNA sample and place in the nebulizer cup:

    DNA50 ug10X TM buffer200 ulsterile glycerol0.5-1 mlsterile ddH2Oq.s.2 ml

  3. Nebulize in an ice-water bath at 30 psi for 2.5 minutes for plasmid, or 8-10 psi for 2.5 minutes for BACs, PACs, fosmids or cosmids.

  4. Briefly centrifuge at 2500 rpm to collect the sample by placing the entire unit in the rotor bucket of a table top centrifuge fitted with pieces of styrofoam to cushion the plastic nebulizer.

  5. Distribute the sample into four 1.5 ml microcentrifuge tubes and ethanol precipitate. Resuspend the dried DNA pellet in 35 ul of 1X TM buffer prior to proceeding with fragment end-repair.

The following persons are acknowledged for contributing methods and suggestions during the assembly of this manual: Stephanie Chissoe, Sandy Clifton, Dennis Burian, Rick Wilson, Din-Pow Ma, James Wong, Leslie Johnston-Dow, Elaine Mardis, Zhili Wang, Kala Iyer, Steve Toth, Goughay Zhang, Hua Qin Pan and other members of the Roe laboratory, both past and present.

5 M NaCl

1X TE Buffer 0.5 M EDTA

C. Random fragment end-repair, size selection, and phosphorylation

Since both sonicated and nebulized DNA fragments usually contain single-stranded ends, the samples are end-repaired prior to ligation into blunt-ended vectors . A combination of T4 DNA polymerase and Klenow DNA polymerase are used to "fill-in" the DNA fragments by catalyzing the 3'-5' incorporation of complementary nucleotides into resultant double-stranded fragments with a 5' overhang. Additionally, the single-stranded 3'-5' exonuclease activity of T4 DNA polymerase is used to degrade 3' overhangs. The reactions included the two enzymes, buffer, and deoxynucleotides and are incubated at 37degC.

Following fragment end-repair, the DNA samples are electrophoresed on a preparative low-melting temperature agarose gel versus the phi-X 174 marker, and after appropriate separation, the fragments in the size range from 1-2Kbp and 2-4Kbp are excised and eluted separately from the gel, as discussed above. Alternatively, the fragments can be purified by fractionation on a Sephacryl S-500 spin column as also discussed above. In both instances, the purified fragments are concentrated by ethanol precipitation followed by resuspension in kinase buffer, and phosphorylation using T4 polynucleotide kinase and rATP. The polynucleotide kinase is removed by phenol extraction and the DNA fragments are concentrated by ethanol precipitation, dried, resuspended in buffer, and ligated into blunt-ended cloning vectors. It should be noted that because a significant portion of nebulized DNA fragments are easily cloned without end-repair or kinase treatment, these two steps can be combined without significantly affecting the overall number of resulting transformed clones .

Protocol

  1. To each tube containing 35 ul of DNA fragments , add:

    0.25 mM dNTPs 2 ulT4 DNA polymerase 3 ul Klenow DNA polymerase2 ul 42 ul

T4 and Klenow DNA polymerases from New England Biolabs.

  1. Incubate at room temperature for 30 minutes.

3a. Add 5 ul of agarose gel loading dye and apply to separate well of a 1% low gel temperature agarose gel and electrophorese for 30-60 minutes at 100-120 mA.

4a. Elute the DNA from each sample lane, ethanol precipitate, and resuspend the dried DNA in 36 ul of sterile ddH2O and add 4 ul of 10X denaturing buffer. There should be five tubes for sonicated fragments and four tubes for nebulized fragments.

5a. Incubate at 70degC for 10 minutes, and place the samples in an ice-water bath.

6a. Add the following reagents for the kinase reaction and incubate at 37 degC for 10-30 minutes:

10 mM rATP 1 ul 10 X kinase buffer 5 ul T4 polynucleotide kinase 1 ul  Final Volume 47 ul

T4 polynucleotide kinase from United States Biochemicals.

7a. Pool the kinase reactions, phenol extract, ethanol precipitate, and resuspend the dried DNA fragments in 40 ul of 10:0.1 TE buffer. This yields a typical concentration of 500-1000 ng/ul.

Alternatively the end-repair and phosphorylation steps can be combined:

1b. Resuspend DNA in 27 ul of 1X TM buffer. Add the following:

10X kinase buffer 5 ul10 mM rATP 5 ul0.25 mM dNTPs 7 ulT4 polynucleotide kinase 1 ul Klenow DNA polymerase 2 ul  T4 DNA polymerase 3 ul  ------------------------------------------------------ Final Volume 50 ul note: if the DNA has been sheared by nebulizing, the T4 DNA polymerase addition here may not be necessary.

2b. Incubate at 37degC for 30 minutes

3b. Add 5 ul of agarose gel loading dye and apply to separate well of a 1% low melting temperature agarose gel and electrophorese for 30-60 minutes at 100-120 mA.

4b. Elute the DNA from each sample lane, ethanol precipitate, resuspend in 10 ul of 10:0.1 TE buffer.

  1. Sambrook, J., Fritsch, E.F., and Maniatis, T., in Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, NY, Vol. 1, 2, 3 .

  2. Glover, D.M. DNA Cloning Volume I: A Practical Approach. IRL Press, Oxford, 1985.

1 M Tris-Cl, pH 6.8

Protein A/G Agarose Proteinase K 10X proteinase K bufferElution Buffer 10 mg/ml Herring Sperm DNA37% Formaldehyde 1.25 M glycineProtocol: For all the following steps, use the pipets that are specifically designated for ChIP use only and the filter pipette tips.To each 10 cm dish of cells, wash plate once with 10ml of PBS, then add 10 ml of Fresh PBS and add 270 ul of 37% formaldehyde, swirl gently to mix, and place at room temp 10 min.

  1. At the end of the incubation, add 1 ml of 1.25 M glycine, swirl to mix.
  2. Aspirate medium
  3. wash plate with 10 ml cold PBS x 2. Aspirate PBS completely after the second wash.
  4. add 500 ul of cold PBS protease inhibitors and scrape cells, collect in a 1.5 ml centrifuge tube. At this point you should pool three plates worth of cells together in the same tube .
  5. centrifuge at 2000 rpm for 2 min at 4 º C.
  6. remove and discard PBS
  7. add 600 ul of ChIP sonication buffer protease inhibitors, and resuspend pellet . Transfer the suspension to a 1.5 ml eppendorf tube.
  8. Place on ice for 10 min.
  9. Sonicate at a setting of 10 or 11 for 15 pulses, 5 seconds per pulse . To eliminate foaming, place the tip of the horn near the bottom of the tube while touching the side with the horn. USE THE PLAIN WHITE TUBES.
  10. Centrifuge at maximal setting at 4 C for 10-15 min.
  11. Remove the supernatant into a fresh tube. This is the Whole Cell Extract , and can be stored at �C80 C at this point, if desired.
  12. Add a sufficient amount of ChIP buffer to perform the immunoprecipitations. A final volume between 1- 1.5 mls is usually good. Add protease inhibitors to this and place on ice.
  13. Add the luciferase plasmid before splitting the samples. One microliter of stock plasmid is good. Also add 10 ml of 100x BSA. Mix well.
  14. Split the samples into and antibody samples of equal volume, making sure you choose an amount that will allow you to withhold 10% of the IP sample volume for your “10% input” samples that you need later for PCR, as well as having a little leftover sample in case you want to examine them later. For example, if you have 1 ml after step 13, split it into a 400 ml sample, a 400ml sample, a 40 ml input sample, with 160 ml leftover.
  15. Label and freeze the 10% Input samples and leftovers.
  16. To the antibody sample, add 5 ul of antibody. This amount may vary according to the antibody used and the size of your sample. To the antibody sample, add nothing.
  17. Place the samples on a nutator in the cold room, and rotate overnight. This step could also be done only for 2hrs in the cold room.
  18. Resuspend Protein A/G agarose so that it forms a uniform suspension. Using a pipet tip with the end clipped off, add 40 ul of this suspension to each immunoprecipitation. Resuspend the protein A/G agarose each time before adding to the next sample, as it settles quickly.
  19. Add 2 ul of a 10 mg/ml solution of herring sperm DNA
  20. Place back on the nutator at 4 C for 1-2 h.
  21. Centrifuge the samples at 4 C for 1 min at 2500 rpm.
  22. Carefully remove the supernatant using a P-1000 and place it in a tube and label it “sample X―sup.” Place this at �C20 C in case you need it later.
  23. Add 1 ml of COLD ChIP buffer , invert the sample to resuspend the resin, and centrifuge for 1 min. at 2500 rpm.
  24. remove and discard the supernatant.
  25. Wash 2X in cold PBS, spinning as above and discarding the supernatants. Note- wash buffer may vary according to conditions, antibody used. See alternate wash buffers at top of protocol.
  26. Add 250 ul of Elution buffer to the resin, and place on a nutator at room temp. for 15-20 min.
  27. Centrifuge at top speed 60s to pellet the resin, remove the supernatant to a fresh tube.
  28. Repeat the elution step , except that it is recommended to place tubes in a 100C heat block for 60s before placing on the nutator.
  29. Spin as in step 29 and combine with the supernatants from step 29.
  30. At this time, add 500 ml of elution buffer to the “10% input samples” from step 16. Process them along with your other samples from here on.
  31. Add 20 ml of 5 M NaCl to each sample, vortex to mix, and place in a 65 C bath for 3-4 h.
  32. Add 1 ml of ROOM TEMP ethanol to each sample place at �C20 C overnight.
  33. Next day, spin the samples at top speed at 4 C for 15-20 min. to pellet the precipitated protein/DNA. Be sure to pre-chill the centrifuge.
  34. Aspirate off the supernatant, add 1 ml of ice cold 70% ethanol, spin again at 4 C for 5 min.
  35. Aspirate off the sup, allow to air dry for 5-10 min.
  36. Dissolve the pellet in 100 ml of TE.
  37. Add 11 ml of 10X Proteinase K buffer, and 1 ul of a 19 mg/ml proteinase K solution.
  38. incubate at 55 C for 1 h.
  39. Add 390 µl TE
  40. Extract w/ 500 µl phenol:CHCl3:isoamylalcohol
    1. Add the stuff
    2. Vortex high speed 1 min
    3. Spin high speed 1 min
  41. Remove top and put into new tube.
  42. Add 44 µl 3 M NaOAc and 1 ml EtOH
  43. Place at �C20C overnight
  44. Spin sample at high speed 4C 5 min.
  45. Aspirate sup. Add 1 ml ice cold 70% EtOH. Spin again high speed 4C 5 min.
  46. Aspirate sup, air dry pellet about 20 min.
  47. Resuspend pellets in 100 µl TE .
  48. Ready for PCR.
  49. If samples, especially the inputs, give strange looking or flat curves on the I-cycler, try adding 390 ml TE to the samples and repeating steps 44-50

D. DNA ligation

DNA ligations are performed by incubating DNA fragments with appropriately linearized cloning vector in the presence of buffer, rATP, and T4 DNA ligase . For random shotgun cloning, sonicated or nebulized fragments are ligated to either SmaI linearized, dephosphorylated double-stranded M13 replicative form or pUC vector by incubation at 4degC overnight. A practical range of concentrations is determined based on the amount of initial DNA, and several different ligations, each with an amount of insert DNA within that range, are used to determine the appropriate insert to vector ratio for the ligation reaction. In addition, several control ligations are performed to test the efficiency of the blunt-ending process, the ligation reaction, and the quality of the vector . These usually included parallel ligations in the absence of insert DNA to determine the background clones arising from self-ligation of inefficiently phosphatased vector. Parallel ligations also are performed with a known blunt-ended insert or insert library, typically an AluI digest of a cosmid, to insure that the blunt-ended ligation reaction would yield sufficient insert containing clones, independent of the repair process.

Protocol

  1. Combine the following reagents in a microcentrifuge tube, and incubate overnight at 4degC:

    DNA fragments 100-1000 ngcloning vector 2 ul 10X ligation buffer 1 ulT4 DNA ligase 1 ul sterile ddH2O q.s.10 ul

The cloning vector typically is SmaI-linearized, CIAP-dephosphorylated pUC vector as several years ago we switched from M13 to pUC-based shotgun cloning. The advantage of obtaining two sequence reads off one isolated shotgun sub-clone seems to outweigh the disadvantage of a few bases less in double-stranded vs single-stranded read lengths. In some instances, including 5% PEG in the ligation reactions also seems to slightly improve the ligation efficiency.

  1. Include control ligation reactions with no insert DNA and with a known blunt-ended insert .

I. General methods

E. Competent cell preparation

There are two main methods for preparation of competent bacterial cells for transformation, the calcium chloride and the electroporation method. For the calcium chloride method, a glycerol cell culture stock of the respective E. coli strain is thawed and added to 50 ml of liquid media. This culture then is preincubated at 37degC for 1 hour, transferred to an incubator-shaker, and is incubated further for 2-3 hours. The cells are pelleted by centrifugation, resuspended in calcium chloride solution, and incubated in an ice-water bath. After another centrifugation step, the resulting cell pellet again is resuspended in calcium chloride to yield the final competent cell suspension. Competent cells are stored at 4degC, for up to several days.

Calcium Chloride Protocol

  1. Thaw a frozen glycerol stock of the appropriate strain of E. coli, add it to an Erlenmeyer flask containing 50 ml of pre-warmed 2xTY media, and pre-incubate in a 37degC water bath for 1 hour with no shaking. Further incubate for 2-3 hours at 37degC with shaking at 250 rpm.

  2. Transfer 40 ml of the cells to a sterile 50 ml polypropylene centrifuge tube, and collect the cells by centrifugation at 3000 rpm for 8 minutes at 4deg C in a GPR centrifuge or 6000 rpm for 8 minutes at 4degC in an RC5-B centrifuge equipped with an SS-34 rotor. For M13-based transformation, save the remaining 10 ml of culture in an ice-water bath for later use.

  3. After centrifugation, decant the supernatant and resuspend the cell pellet in one-half volume of cold, sterile 50 mM calcium chloride, incubate in an ice-water bath for 20 minutes, and centrifuge as before.

  4. Decant the supernatant and gently resuspend the cell pellet in one-tenth volume of cold, sterile 50 mM calcium chloride to yield the final competent cell suspension.

Preparation of calcium chloride competent cells for frozen storage

  1. Transfer 166 ul of the competent cell suspension to sterile Falcon culture tubes.

  2. Add 34 ul of sterile 100% glycerol to the 166 ul aliquots of the final competent cell suspension prepared above, giving a final concentration of 17 % glycerol.

  3. The competent cells then should be placed at -70degC and can be stored indefinately.

  4. To use competent cells for transformation, remove from freezer and thaw for a few minutes at 37degC. Place on ice, add plasmid DNA and incubate for one hour as in the standard transformation procedure. Then heat shock at 42degC for 2 minutes, cool briefly, add 1 ml of 2xTY and incubate for 1 hour at 37degC before spreading on plates.

Electroporation Protocol

Preparation of Electro-competent Cells:

  1. Grow XL1-Blue cells on a tetracycline plate

  2. Inoculate 3 ml of YENB and grow overnight at 37 degrees C with shaking at 250 rpm in the New Brunswick incubator shaker.

  3. Inoculate the 3 ml of overnight growth into 1 liter of YENB and grow to an A600 of 0.5 .

  4. Centrifuge at 5000 rpm at 4 degrees C for 10 minutes in the Sorval GS-3 Rotor.

  5. Resuspend each of the two pellets in 100 ml of ice cold sterile double distilled water and combine the resuspended pellets into one Sorval centrifuge bottle and centrifuge at 5000 rpm at 4 degrees C for 10 minutes in the Sorval GS-3 Rotor once more. Note: The purpose of all these centrifugation/resuspension/centrifugation steps is to insure that the cells are essentially "salt-free" as salt causes arching during the electroporation step.

  6. Resuspend the pellet in 100 ml of 10% ice cold sterile glycerol, centrifuge as above, and finally resuspend the pellet in 2 ml of 10% ice cold sterile glycerol to give salt-free, concentrated electrocompetent cells.

  7. Aliquote 40 ul of these electrocompetent cells into small snap cap tubes and immediately freeze by placing in curshed dry ice and then store at -70 degrees C until needed.

Electroporation Protocol for transformations using double-stranded plasmids

  1. Thaw the electro-competent cells on ice for about one minute.

  2. Add 2-3 ul of the ligation mix to the cells.

  3. transfer 40 ul of the cells into to BTX Electroporation cuvettes PLUS and MAKE SURE THAT THE CELLS COVER THE BOTTOM OF THE CUVETTE.

  4. Turn on the Bio Rad E. coli Pulser and set the current to 2.5 KV by pushing the "Lower" and "Raise" bottoms simultaneously twice.

  5. Place the cuvette in the holder and slide it into position.

  6. Charge by pressing the "Charge" bottom until you hear the beep.

  7. Immediately, suspend the cells in 1 ml of YENB and transfer into a Falcon tube.

  8. Incubate the cells at 37 degrees C for 30 minutes at 250 rpm shaker.

  9. Spin the cells in BECKMAN table-top centrifuge for 8 minutes at 2500 rpm

  10. Resuspend the cells in 200 ul fresh YENB and add 30 ul of 20 mg/ml XGAL and 30 ul of 25 mg/ml IPTG

  11. Plate ~130 ul of the cells on pre-warmed LB-amp plates.

Reference:Rakesh C. Sharma and Robert T. Schimke, "Preparation of Electro-competent E. coli Using Salt-free Growth Medium", Biotechniques 20, 42-44 .

A. Phenol extraction of DNA samples

Phenol extraction is a common technique used to purify a DNA sample . Typically, an equal volume of TE-saturated phenol is added to an aqueous DNA sample in a microcentrifuge tube. The mixture is vigorously vortexed, and then centrifuged to enact phase separation. The upper, aqueous layer carefully is removed to a new tube, avoiding the phenol interface and then is subjected to two ether extractions to remove residual phenol. An equal volume of water-saturated ether is added to the tube, the mixture is vortexed, and the tube is centrifuged to allow phase separation. The upper, ether layer is removed and discarded, including phenol droplets at the interface. After this extraction is repeated, the DNA is concentrated by ethanol precipitation.

Protocol

  1. Add an equal volume of TE-saturated phenol to the DNA sample contained in a 1.5 ml microcentrifuge tube and vortex for 15-30 seconds.

  2. Centrifuge the sample for 5 minutes at room temperature to separate the phases.

  3. Remove about 90% of the upper, aqueous layer to a clean tube, carefully avoiding proteins at the aqueous:phenol interface. At this stage the aqueous phase can be extracted a second time with an equal volume of 1:1 TE-saturated phenol:chloroform, centrifuged and removed to a clean tube as above but this additional extraction usually is not necessary if care is taken during the first phenol extraction.

  4. Add an equal volume of water-saturated ether, vortex briefly, and centrifuge for 3 minutes at room temperature. Remove and discard the upper, ether layer, taking care to remove phenol droplets at the ether:aqueous interface. Repeat the ether extraction.

  5. Ethanol precipitate the DNA by adding 2.5-3 volumes of ethanol-acetate, as discussed below.

F. Calcium Chloride treated bacterial cell transformation

A brief background discussion of transformation and transfection can be found in the Appendix.

For DNA transformation , the entire DNA ligation reaction is added to an aliquot of competent cells, which is mixed gently, and incubated in an ice-water bath. This mixture then is heat-shocked briefly in a 42degC water bath for 2-5 minutes. At this point in the transformation, the method varied slightly depending on whether the cloning vector is M13-based or pUC-based.

For M13-based transformation , an aliquot of non-competent cells is added to the heat-shocked mixture, as is the lac operon inducer homologue, IPTG, and the b-galactosidase chromogenic substrate, x-gal. Melted top agar is added, and the transformation mixture then is poured onto the surface of an agar plate. After the top agar solidified, the plates are inverted and incubated overnight at 37degC.

For pUC-based transformation , an aliquot of liquid media is added to the heat-shocked mixture, which then is incubated in a 37degC water bath for 15-20 minutes. After recovery, the cell suspension is concentrated by centrifugation and then gently resuspended in a smaller volume of fresh liquid media. IPTG and x-gal are added to the cell mixture, which is spread onto the surface of an ampicillin-containing agar plate. After the cell mixture had diffused into the agar medium, the plates are inverted and incubated overnight at 37degC.

Protocol

  1. Add the entire ligation reaction to a 12 X 75 Falcon tube containing 0.2-0.3 ml of competent cells, mix gently, and incubate in an ice-water bath for 40-60 minutes. .

  2. Heat shock the cells by incubation at 42degC for 2-5 minutes.

For M13-based transformation:

3a. Add the following reagents to the heat shocked transformation mixture:

Non-competent cells0.2 mlIPTG  25 ulx-gal  25 ullambda top agar2.5 ml

4a. Mix by briefly vortexing, and then quickly pour onto the surface of a pre-warmed lambda agar plate.

5a. Allow 10-20 minutes for the agar to harden, and then invert and incubate overnight at 37degC.

For pUC-based transformation:

3b. Add the following reagents to the heat shocked transformation mixture, add 1 ml of fresh 2xTY and incubate in a 37degC water bath for 15-30 minutes.

4b. Collect the cells by centrifugation at 3000 rpm for 5 minutes, decant the supernatant, and gently resuspend in 0.2 ml of fresh 2xTY.

5b. Add 25 ul IPTG and 25 ul x-gal , mix and pour onto the surface of a pre-warmed LB-Amp plate. Spread over the agar surface using a sterile bent glass rod or sterile inoculating loop.

6b. Allow 10-20 minutes for the liquid to diffuse into the agar, and then invert and incubate overnight at 37degC.

For pBR322, pAT153 or other non-lacZ containing vectors:

3b. Add 1 ml of fresh 2xTY to the cells and incubate for 15-30 minutes at 37 degC. Spread approximately 50 ul on L plates containing antibiotic using a sterile glass spreader. Incubate the plates overnight at 37degC.

B. Concentration of DNA by ethanol precipitation

Typically, 2.5 - 3 volumes of an ethanol/acetate solution is added to the DNA sample in a microcentrifuge tube, which is placed in an ice-water bath for at least 10 minutes. Frequently, this precipitation is performed by incubation at -20C overnight . To recover the precipitated DNA, the tube is centrifuged, the supernatant discarded, and the DNA pellet is rinsed with a more dilute ethanol solution. After a second centrifugation, the supernatant again is discarded, and the DNA pellet is dried in a Speedy-Vac.

Protocol

  1. Add 2.5-3 volumes of 95% ethanol/0.12 M sodium acetate to the DNA sample contained in a 1.5 ml microcentrifuge tube, invert to mix, and incubate in an ice-water bath for at least 10 minutes. It is possible to place the sample at -20degC overnight at this stage.

  2. Centrifuge at 12,000 rpm in a microcentrifuge for 15 minutes at 4 degC, decant the supernatant, and drain inverted on a paper towel.

  3. Add 80% ethanol , incubate at room temperature for 5-10 minutes and centrifuge again for 5 minutes, and decant and drain the tube, as above.

  4. Place the tube in a Savant Speed-Vac and dry the DNA pellet for about 5-10 minutes, or until dry.

  5. Always dissolve dried DNA in 10 mM Tris-HCl, pH 7.6-8.0, 0.1 mM EDTA .

  6. It is advisable to aliquot the DNA purified in large scale isolations into several small microcentrifuge tubes for frozen storage because repeated freezing and thawing is not advisable.

Notes on precipitation of nucleic acids

A. General rules

Most nucleic acids may be precipitated by addition of monovalent cations and two to three volumes of cold 95% ethanol, followed by incubation at 0 to -70 degC. The DNA or RNA then may be pelleted by centrifugation at 10 to 13,000 x g. for 15 minutes at 4degC. A subsequent wash with 70% ethanol, followed by brief centrifugation, removes residual salt and moisture.

The general procedure for precipitating DNA and RNA is:

  1. Add one-tenth volume of 3M NaOAc, pH 5.2* to the nucleic acid solution to be precipitated,

  2. Add two volumes of cold 95% ethanol,

  3. Place at -70degC for at least 30 minutes, or at -20degC overnight.

or alternatively

  1. Combine 95 ml of 100% ethanol with 4 ml of 3 M NaOAc and 1ml of sterile water. Mix by inversion and store at -20degC.

  2. Add 2.5 volumes of cold ethanol/acetate solution to the nucleic acid solution to be precipitated.

  3. Place at at -70degC for at least 30 minutes or -20degC for two hours to overnight.

* 5M NH4OAc, pH 7.4, NaCl and LiCl may be used as alternatives to NaOAc. DNA also may be precipitated by addition of 0.6 volumes of isopropanol.

B. Oligonucleotides

Add one-tenth volume of 3M NaOAc, pH 6.5, and three volumes of cold 95% ethanol.

Place at -70degC for at least one hour.

C. RNA

Add one-tenth volume of 1M NaOAc, pH 4.5, and 2.5 volumes of cold 95% ethanol.

Precipitate large volumes at -20degC overnight.

Small volume samples may be precipitated by placing in powdered dry ice or dry ice-ethanol bath for five to 10 minutes.

D. Isobutanol concentration of DNA

DNA samples may be concentrated by extraction with isobutanol. Add slightly more than one volume of isobutanol, vortex vigorously and centrifuge to separate the phases. Discard the isobutanol phase, and extract once with water-saturated diethyl ether to remove residual isobutanol. The nucleic acid then may be ethanol precipitated as described above.

E. Notes on phenol extraction of nucleic acids

The standard and preferred way to remove proteins from nucleic acid solutions is by extraction with neutralized phenol or phenol/chloroform. Generally, samples are extracted by addition of one-half volume of neutralized phenol to the sample, followed by vigorous mixing for a few seconds to form an emulsion. Following centrifugation for a few minutes, the aqueous phase containing the nucleic acid is recovered and transferred to a clean tube. Residual phenol then is removed by extraction with an equal volume of water-saturated diethyl ether. Following centrifugation to separate the phases, the ether phase is discarded and the nucleic acid is ethanol precipitated as described above.

A 1:1 mixture of phenol and chloroform also is useful for the removal of protein from nucleic acid samples. Following extraction with phenol/chloroform, the sample should be extracted once with an equal volume of chloroform, and ethanol precipitated as described above.

G. Microcentrifuge Tube Transformation

Microcentrifuge transformations are recommended when a single plasmid is being retransformed or for qualitative transformation experiments. Shotgun cloning experiments should be transformed using the large scale transformation, since the objective is to efficiently obtain transformation of hundreds of distinct recombinant plasmids.

  1. Inoculate 50 ml of fresh 2xTY media with 3 to 5 ml of a fresh overnight culture of a suitable host strain and incubate for 2 to 3 hours at 37deg C.

  2. Transfer 1 ml of the culture into a 1.5 ml tube and centrifuge for 5 min at room temperature. Use 1 tube of culture per DNA sample to be transformed.

  3. Decant supernatant, and resuspend the cell pellet in 500 ul of sterile, cold 50 mM calcium chloride. Gently vortex if necessary.

  4. Incubate 5 min. on ice.

  5. Centrifuge as before, decant and resuspend the competent cell pellet in 100 ul of calcium chloride.

  6. Transfer each 100 ul sample of competent cells to chilled 12 x 75 mm Falcon tubes which contain 3 to 5 ul of DNA sample .

  7. Incubate on ice for 15 minutes.

  8. Heat shock the sample at 42degC for 5 minutes.

  9. Add 1 ml of fresh 2xTY to each sample and recover the cells by incubating at 37degC for 15 min.

  10. For lacZ containing vectors add 25 ul of 20 mg/ml IPTG and 25 ul of 24 mg/ml X-Gal .

  11. Add 2.5 ml of soft top agar to each sample, vortex and quickly pour onto the surface of a TYE-AMP agar plate. Allow at least 15-30 min. for the agar to solidify.

  12. Invert the plates and incubate overnight at 37degC.

C. Restriction digestion

Restriction enzyme digestions are performed by incubating double-stranded DNA molecules with an appropriate amount of restriction enzyme, in its respective buffer as recommended by the supplier, and at the optimal temperature for that specific enzyme. The optimal sodium chloride concentration in the reaction varies for different enzymes, and a set of three standard buffers containing three concentrations of sodium chloride are prepared and used when necessary. Typical digestions included a unit of enzyme per microgram of starting DNA, and one enzyme unit usually is defined as the amount of enzyme needed to completely digest one microgram of double-stranded DNA in one hour at the appropriate temperature. These reactions usually are incubated for 1-3 hours, to insure complete digestion, at the optimal temperature for enzyme activity, typically 37degC. See the Appendix for a listing of restriction sites present in the M13 MCS and a listing of various restriction enzymes, incubation conditions and cut sites.

Protocol

  1. Prepare the reaction for restriction digestion by adding the following reagents in the order listed to a microcentrifuge tube:

    皇家赌场号hj85, sterile ddH20 q.s 10X assay buffer one-tenth volumeDNA x ulrestriction enzyme* y ul Total volume z ul

*If desired, more than one enzyme can be included in the digest if both enzymes are active in the same buffer and the same incubation temperature.

Note: The volume of the reaction depends on the amount and size of the DNA being digested. Larger DNAs should be digested in larger total volumes , as should greater amounts of DNA.

Refer to the vendor's catalog for the chart of enzyme activity in a range of salt concentrations to choose the appropriate assay buffer . Restriction enzymes are purchased from Bethesda Research Laboratories, New England Biolabs, or United States Biochemicals.

  1. Gently mix by pipetting and incubate the reaction at the appropriate temperature for 1-3 hours.

  2. Inactivate the enzyme by heating at 70-100degC for 10 minutes or by phenol extraction . Prior to use in further protocols such as dephosphorylation or ligation, an aliquot of the digestion should be assayed by agarose gel electrophoresis versus non-digested DNA and a size marker, if necessary.

D. Agarose gel electrophoresis

Agarose gel electrophoresis is employed to check the progression of a restriction enzyme digestion, to quickly determine the yield and purity of a DNA isolation or PCR reaction, and to size fractionate DNA molecules, which then could be eluted from the gel. Prior to gel casting, dried agarose is dissolved in buffer by heating and the warm gel solution then is poured into a mold , which is fitted with a well-forming comb. The percentage of agarose in the gel varied. Although 0.7% agarose gels typically are used, in cases where the accurate size fractionation of DNA molecules smaller than 1 kb is required, a 1, 1.5, or 2% agarose gel is prepared, depending on the expected size of the fragment. Ethidium bromide is included in the gel matrix to enable fluorescent visualization of the DNA fragments under UV light. Agarose gels are submerged in electrophoresis buffer in a horizontal electrophoresis apparatus. The DNA samples are mixed with gel tracking dye and loaded into the sample wells. Electrophoresis usually is at 150

  • 200 mA for 0.5-1 hour at room temperature, depending on the desired separation. When low-melting agarose is used for preparative agarose gels, electrophoresis is at 100-120 mA for 0.5-1 hour, again depending on the desired separation, and a fan is positioned such that the heat generated is rapidly dissipated. Size markers are co-electrophoresed with DNA samples, when appropriate for fragment size determination. Two size markers are used, phi-X 174 cleaved with restriction endonuclease HaeIII to identify fragments between 0.3-2 kb and lambda phage cleaved with restriction endonuclease HindIII to identify fragments between 2-23 kb. After electrophoresis, the gel is placed on a UV light box and a picture of the fluorescent ethidium bromide-stained DNA separation pattern is taken with a Polaroid camera.

Protocol

  1. Prepare an agarose gel, according to recipes listed below, by combining the agarose and water in a 500 ml Ehrlenmeyer flask, and heating in a microwave for 2-4 minutes until the agarose is dissolved.

    0.7% 1.0% 2.0% agarose 1.05 g 1.5 g 3.0 g 20X TAE 7.5 ml 7.5 ml 7.5 ml ddH2O 142.5 ml 142.5 ml 142.5 ml EtBr 25 ul 25 ul 25 ul total vol 150 ml 150 ml 150 ml

Genetic technology grade or low gel temperature agarose from Schwarz/Mann Biotech.

  1. Add 20X TAE and ethidium bromide , swirl to mix, and pour the gel onto a taped plate with casting combs in place. Allow 20-30 minutes for solidification.

  2. Carefully remove the tape and the gel casting combs and place the gel in a horizontal electrophoresis apparatus. Add 1X TAE electrophoresis buffer to the reservoirs until the buffer just covers the agarose gel.

  3. Add at least one-tenth volume of 10X agarose gel loading dye to each DNA sample, mix, and load into the wells. Electrophorese the gel at 150-200 mA until the required separation has been achieved, usually 0.5-1 hour , and cool the gel during electrophoresis with a fan. Visualize the DNA fragments on a long wave UV light box and photograph with a Polaroid camera.

E. Elution of DNA fragments from agarose

DNA fragments are eluted from low-melting temperature agarose gels using an unpublished procedure first developed by Dr. Roe. Here, the band of interest is excised with a sterile razor blade, placed in a microcentrifuge tube, frozen at -70degC, and then melted. Then, TE-saturated phenol is added to the melted gel slice, and the mixture again is frozen and then thawed. After this second thawing, the tube is centrifuged and the aqueous layer removed to a new tube. Residual phenol is removed with two ether extractions, and the DNA is concentrated by ethanol precipitation.

Protocol

  1. Place excised DNA-containing agarose gel slice in a 1.5 ml microcentrifuge tube and freeze at -70degC for at least 15 minutes, or until frozen. It is possible to pause at this stage in the elution procedure and leave the gel slice frozen at -70degC.

  2. Melt the slice by incubating the tube at 65degC.

  3. Add one-volume of TE-saturated phenol, vortex for 30 seconds, and freeze the sample at -70degC for 15 minutes.

  4. Thaw the sample, and centrifuge in a microcentrifuge at 12,000 rpm for 5 minutes at room temperature to separate the phases. The aqueous phase then is removed to a clean tube, extracted twice with equal volume ether, ethanol precipitated, and the DNA pellet is rinsed and dried.

F. Kinase end-labeling of DNA

Typical 5'-kinase labeling reactions included the DNA to be labeled, [[gamma]]-32-P-rATP, T4 polynucleotide kinase, and buffer . After incubation at 37degC, reactions are heat inactivated by incubation at 80degC. Portions of the reactions are mixed with gel loading dye and loaded into a well of a polyacrylamide gel and electrophoresed. The gel percentage and electrophoresis conditions varied depending on the sizes of the DNA molecules of interest. After electrophoresis, the gel is dried and exposed to x-ray film, as discussed below for radiolabeled DNA sequencing.

Protocol

  1. Add the following reagents to a 0.5 ml microcentrifuge tube, in the order listed:

    sterile ddH2Oq.s 10X kinase buffer1 ulDNAx ul[[gamma]]-[32-P]-rATP10 uCiT4 polynucleotide kinase1 ul 10 ul

[[gamma]]-[32-P]-rATP ICN and T4 polynucleotide kinase from United States Biochemicals.

  1. Incubate at 37degC for 30-60 minutes.

  2. Heat the reaction at 65degC for 10 minutes to inactivate the kinase.

G. Bacterial cell maintenance

Four strains of E. coli are used in these studies: JM101 for M13 infection and isolation , XL1BMRF' for M13 or pUC-based DNA transformation , and ED8767 for cosmid DNA transformation . To maintain their respective F' episomes necessary for M13 viral infection , JM101 is streaked onto a M9 minimal media LB-TetJM101M9ED8767LB

  1. Pick several colonies into a 12 X 75 mm Falcon tube containing a 2 ml aliquot of the respective liquid media, and incubate for 8-10 hours at 37degC with shaking at 250 rpm.

  2. Transfer the 2 ml culture into an Ehrlenmeyer flask containing 50 ml of the respective liquid media and further incubate overnight at 37degC with shaking at 250 rpm.

  3. Add 12.5 ml of sterile glycerol for a final concentration of 20%, and distribute the culture in 1.3 ml aliquots into 12 X 75 mm Falcon tubes.

  4. Store glycerol cell stocks frozen at -70degC until use.

Notes on Restriction/Modification Bacterial Strains:

  1. EcoK -hsdRMS genes=attack DNA not protected by adenine methylation. .

  2. mcrA , mcrBC, and mrr=methylation requiring systems that attack DNA only when it IS methylated

  3. In general, it is best to use a strain lacking Mcr and Mrr systems when cloning genomic DNA from an organism with methylcytosine such as mammals, higher plants , and many prokaryotes.

  4. The use of D hosts=general methylation tolerance and suitability for clones with N6 methyladenine as well as 5mC .

  5. XL1-Blue MRF'=D182, D172,endA1, supE44, thi-1, recA, gyrA96, relA1, lac, l-, [F' proAB, lacIqZDM15, Tn10].

Host Mutation Descriptions:

araInability to utilize arabinose.deoRRegulatory gene that allows for constitutive synthesis for genes involved in deoxyribose synthesis. Allows for the uptake of large plasmids. endADNA specific endonuclease I. Mutation shown to improve yield and quality of DNA from plasmid minipreps.F'F' episome, male E. coli host. Necessary for M13 infection.galKInability to utilize galactose.galTInability to utilize galactose.gyrAMutation in DNA gyrase. Confers resistance to nalidixic acid.hflHigh frequency of lysogeny. Mutation increases lambda lysogeny by inactivating specific protease.lacI Repressor protein of lac operon. LacIq is a mutant lacI that overproduces the repressor protein.lacYLactose utilization; galactosidase permease .lacZb-D-galactosidase; lactose utilization. Cells with lacZ mutations produce white colonies in the presence of X-gal; wild type produce blue colonies.lacZdM15A specific N-terminal deletion which permits the a-complementation segment present on a phagemid or plasmid vector to make functional lacZ protein.DlonDeletion of the lon protease. Reduces degradation of b-galactosidase fusion proteins to enhance antibody screening of l libraries.malAInability to utilize maltose.proABMutants require proline for growth in minimal media.recAGene central to general recombination and DNA repair. Mutation eliminates general recombination and renders bacteria sensitive to UV light.rec BCDExonuclease V. Mutation in recB or recC reduces general recombination to a hundredth of its normal level and affects DNA repair.relARelaxed phenotype; permits RNA synthesis in the absence of protein synthesis.rspL30S ribosomal sub-unit protein S12. Mutation makes cells resistant to streptomycin. Also written strA. recJExonuclease involved in alternate recombination pathways of E. coli.strASee rspL.sbcBCExonuclease I. Permits general recombination in recBC mutants.supESupressor of amber  mutations. Some phage require a mutation in this gene in order to grow.supFSupressor of amber  mutations. Some phage require a mutation in this gene in order to grow.thi-1Mutants require vitamin B1 for growth on minimal media.traD36mutation inactivates conjugal transfer of F' episome.umuCComponent of SOS repair pathway.uvrCComponent of UV excision pathway.xylAInability to utilize xylose.damDNA adenine methylase/ Mutation blocks methylation of Adenine residues in the recognition sequence 5'-G*ATC-3' dcmDNA cytosine methylase/Mutation blocks methylation of cytosine residues in the recognition sequences 5'-C*CAGG-3' or 5'-C*CTGG-3' hsdME. coli methylase/ Mutation blocks sequence specific methylation AN6*ACNNNNNNGTGC or GCN6*ACNNNNNNGTT . DNA isloated from a HsdM- strain will be restricted by a HsdR  host.hsd R17Restriction negative and modification positive. Allows cloning of DNA without cleavage by endogenous restriction endonucleases. DNA prepared from hosts with this marker can efficiently transform rK  E. coli hosts.hsdS20Restriction negative and modification negative.Allows cloning of DNA without cleavage by endogenous restriction endonucleases . DNA prepared from hosts with this marker is unmethylated by the hsdS20 modificationsystem.mcrAE. coli restriction system/ Mutation prevents McrA restriction of methylated DNA of sequence 5'-C*CGG .mcrCBE. coli restriction system/ Mutation prevents McrCB restriction of methylated DNA of sequence 5'-G5*C, 5'-G5h*C, or 5'-GN4*C .mrrE. coli restriction system/ Mutation prevents Mrr restriction of methylated DNA of sequence 5'-G*AC or 5'-C*AG . Mutation also prevents McrF restriction of methylated cytosine sequences.

Other Descriptions:

cmrChloramphenicol resistancekanrKanamycin resistancetetrTetracycline resistancestrrStreptomycin resistanceDIndicates a deletion of genes following it.Tn10A transposon that normally codes for tetrTn5A transposon that normally codes for kanrspi-Refers to red-gam- mutant derivatives of lambda defined by their ability to form plaques on E. coli P2 lysogens.Commonly used bacterial strainsC600- F-, e14, mcrA, thr-1 supE44, thi-1, leuB6, lacY1, tonA21, l--for plating lambda  libraries, grows well in L broth, 2x TY, plate on NZYDT Mg.-Huynh, Young, and Davis  DNA Cloning, Vol. 1, 56-110.DH1- F-, recA1, endA1, gyrA96, thi-1, hsdR17 , supE44, relA1, l--for plasmid transformation, grows well on L broth and plates.-Hanahan  J. Mol. Biol. 166, 557-580.XL1Blue-MRF' - D182, D172,endA1, supE44, thi-1, recA, gyrA96, relA1, lac, l-, [F'proAB, lac IqZDM15, Tn10 ] -For plating or glycerol stocks, grow in LB with 20 mg/ml of tetracycline. For transfection, grow in tryptone broth containing 10 mM MgSO4 and 0.2% maltose.  For picking plaques, grow glycerol stock in LB to an O.D. of 0.5 at 600 nm . When at 0.5, add MgSO4 to a final concentration of 10 mM.SURE Cells - Stratagene - e14, D171, sbcC, recB, recJ, umuC::Tn5 , uvrC, supE44, lac, gyrA96, relA1, thi-1, end A1[F'proAB, lacIqDM15, Tn10]. An uncharacterized mutation enhances the a - complementation to give a more intense blue color on plates containing X-gal and IPTG.

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