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ET重组的方法皇家赌场号hj85

文章作者:生命科学 上传时间:2019-08-27

核心提示:Oligo designThe 5' end - choose 42 or more nts for the homology a

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

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

  • The 5' end - choose 42 or more nts for the homology arms from the target DNA sequence simply according to where you want to insert the PCR fragment. .
  • The 3' end - choose about 18-30 nt for the PCR primer region as you would for any other PCR. Use of the Oligo 4 software program can help, especially for shorter primers.
  • Between these two regions, you can stick whatever you like - within the limits of oligo synthesis. We routinely put loxP or FRTs here, for subsequent recombination by Cre or FLP recombinases respectively.

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

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.

PCR reaction and recipient plasmid DNA preparation

  • The oligo powder was dissolved in 500 ul of dH2O. Extract oligo solution with phenol/chloroform as follows: 100 ul oligo solution 12 ul 3M NaAc 120 ul phenol:CHCl3 Vortex for 30', spin for 2-3 min, add 360 ul ethanol, place in -80oC freezer for 5-10 min, spin for 5 min, wash once with 70% ethanol, dry under vacuum and dissolve in 100 ul of dH2O.
  • PCR reaction 33 ul dH2O 5 ul 10 x PCR reaction buffer 5 ul 2.5 mM dNTP 1.5 ul upper oligo 1.5 ul lower oligo 2 ul templat 0.5 ul Taq polymerase Annealing temperature is important, usually 60oC - 62oC is optimal.
  • Purify the PCR products by Qiagen column and elute with 2x 50 ul dH2O.
  • Add 10x Dpn 1 buffer and 2 ul Dpn 1 and digest for 1 hour.
  • Extract with Phenol:CHCl3 once, precipitate with ethanol as above and redissolve in 5 ul dH2O
  • If competent cells harbour recipient plasmid, 0.3 ug of PCR product will be used for transformation.
  • For co-transformation, extract recipient DNA once with phenol:CHCl3 as above and dissolve in dH2O at 1 ug/ul. Mix PCR products with recipient plasmid DNA . 1 ul of mixture will be used for transformation.

5 M NaCl

1X TE Buffer 0.5 M EDTA

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.

Preparation of competent bacterial cells

  • Transform your host with the ET expression plasmid you are using by standard procedures and plate.
  • Pick single colony and grow in 5 ml LB medium overnight.
  • Transfer 0.7ml into 70 ml of LB medium and grow them at 37oC with shaking.
  • Prepare 10% glycerol with dH2O, and cool down on ice for at least 3 hours before using.
  • When the cells reach OD600 = 0.1-0,15, add 0.7ml 10% L-arabinose to induce ET protein expression.
  • After a further 45-60 minutes, the cells should be at OD600 of 0.3-0.4. Harvest.
  • make sure the centrifuge and SA600 or SS34 rotor is very cold by centrifuging for 10 min, -5oC at 4,000 rpm.
  • Spin 35 mls cells for 10 min at 7,000 rpm at -5oC. Put the other 35 mls on ice.
  • Pour away the supernatant, add the second 35 mls and respin.
  • Pour away supernatant, put tube on ice, resuspend cells in 5 ml ice cold 10% glycerol with an ice cold 5ml pipette. Add a further 25 mls and centrigue.
  • Repeat the above step twice.
  • Pour away supernatant and immediately dry the tube out with Kleenex tissue takning care not to touch the pellet.
  • Resuspend the cells in the remaining liquid .
  • Transfer 50 ul of cells into each pre-cooled eppendorf tube and freeze in liquid N2 or use immediately. All the steps should be done as cold as possible, always on ice! The glass pipettes should be cooled once or twice by pipetting cold 10% glycerol up and down before pipetting cells.

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

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.

ET重组的方法皇家赌场号hj85。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.

Transformation

  • Wash cuvettes at least 10 times with dH2O, precool them on ice for least 5 min.
  • Thaw competent cells on ice and add 1 ul of PCR product or 1 ul mixture of PCR plus DNA for co-transformation.
  • Electroporate the cells at 2.3 kV , 25 uF with Pulse controller set to 200 ohms)
  • Add 1 ml of LB medium and transfer back into the eppendorf tube.
  • Incubate at 37oC for 1 to 1.5 hours with shaking.
  • Plate 100ul on suitable antibiotoc plates, spin the rest, resuspend in 100ul and plate that..

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 .

Using Cre or Flp transient plasmids

705-Cre and 705-Flp plasmids are based on the pSC101 temperature sensitive origin. This origin maintains a low copy number and replicates at 30oC. These plasmids will be lost from cells when they are incubated at temperatures above 37oC. In addition, Cre and Flp are expressed from the lambdaPR promoter, which expresses weakly at 30oC and strongly at 37oC. Thus 705-Cre and 705-Flp can be used to give a transient burst of expression after which they will be eliminated so the recombined product can be isolated uncontaminated by their presence.

  • Transform the E.coli host containing the plasmid to be site specifically recombined with 705-Flp or 705-Cre. .
  • Incubate at 30oC for 1.5 hr with shaking after transformation.
  • Plate on L.B. plates containing 25 ug/ml of chloramphenicol .
  • Incubate at 30oC for 2 days.
  • Pick single colonies and grow a mini-prep in L.B. medium overnight at 37-38oC .
  • Streak onto L.B. agar plate without chloramphenicol, and culture at 37oC.
  • Pick single colonies and streak on Cm plates to check if the 705 plasmid is lost.

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 .

Using Cre or Flp expressing E.coli

294-Cre and 294-Flp were generated by integrating 705-Cre and 705-Flp into the lacZ locus of E.coli strain, MM294

Check the plasmid by mini-prep to see the loxP or FRT sites lost or not.

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.

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. 皇家赌场号hj85,Invert the plates and incubate overnight at 37degC.

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