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12b Plasmid 2
3723 F02
Biochemistry 3723 Lecture 12b Plasmids #2Nov. 13, 2002
I.Plasmid purification: Separate from chromosomal DNA, RNA, & protein
A.Grow from single cell (clone) to get one type-one plasmid with one insert. Statistically, only one plasmid per cell.
1.Start from well-isolated colony on plate
2.Relaxed plasmid 20-200 copies/cell
a.Grow to late log phase (12-16 hrs) in rich medium with high conc. of Amp: LB or Terrific Broth, to get high plasmid yield.
b.LB contains Tryptone, Yeast Extract, NaCl while Terrific Broth contains Tryptone, YE, KPi, glycerol.
4.Centrifuge to collect cells: Cells from 1–1.5 ml enough for miniprep, such as we do today. Useful when you want to isolate just enough DNA to analyze by restriction digestion and gel electrophoresis. With current technology, enough to sequence if good quality. We do more (4.5 ml) to increase yield.
B.Lyse Cells
1.Considerations: Need intact plasmid: supercoil, not sheared or nicked
2.Method of cell lysis depends on plasmid size
a.>15 kb must be very gentle to avoid sheer--two steps:
i.iso-osmotic: sucrose + lysozyme + EDTA spheroplasts
ii.then add SDS to gently lyse cells
b.smaller plasmids: not so fussy -- use alkaline lysis--"old fashioned" way
i.Add glucose, buffer, EDTA, (some people add lysozyme),
ii.then SDS and either boil or add NaOH
c.We use QIAprep Alkaline SDS prep as follows:
i.Buffer P1: Tris, EDTA, RNase. EDTA to chelate Mg++ to inactivate any DNAses present in cells, RNase to degrade RNA
ii .Buffer P2 = NaOH/SDS (freshly prepared): SDS dissolves cell walls and denatures proteins. NaOH helps denature proteins and disrupts base pairing of DNA to denature. Must prepare fresh or keep tightly sealed so CO2 doesn't neutralize NaOH
C.Separate plasmid DNA from other DNA, and protein by differential precipitation and selective chromatography (Qiagen columns)
1.Precipitation of Proteins: Solution N3 (Proprietary--contains chiatropic agent to denature and precipitate proteins). Chromosomal DNA because of large size precipitates in complex with protein. Centrifuge to remove protein and much of chromosomal DNA. Plasmid DNA remains in solution.
2.Column or membrane--
a.For mini-prep Qiagen uses "unique silica membrane for selective adsorption of plasmid DNA in high-salt buffer and elution in low salt buffer."
b.For larger scale prep --ion exchange resin (see diagram below). Bind DNA in low salt and elute with high salt. Then precipitate plasmid DNA with isopropyl alcohol to concentrate.
3.There may be times when you would want to use another method, such as CsCl density gradient centrifugation, but these are time-consuming and tedious.
B.Technical on Isolation of DNA
1.Watch volumes, mixing (vortexing will shear DNA), timing.
2.At step 5, wait until all are ready so all use centrifuge together.
3.Be sure to save your DNA, properly labeled!
III. Restriction of DNA
A.At this point have, hopefully, recombinant plasmid carrying mystery DNA. To analyze insert, do restriction analysis with EcoRI & SacI
B.Conditions
1.Use MultiCore buffer, which works for both enzymes used. (Different enyzmes have different requirements of buffer, salt, etc.)
2.Do undigested control, two single digests and double digest on each plasmid
3.Judy and I will stop: Stop solution, which is also gel-loading buffer, contains
a.glycerol to make dense, needed when load gels
b.EDTA to protect from DNAses,
c.BPB is tracking dye for agarose gel
IV. Electrophoretic separation of DNA fragments.
A.Principles
1.Agarose gels: Standard method to separate, identify, purify DNA fragments
a.simple, rapid, high resolution
i.0.200-50 kb in various agarose concentrations
ii.Up to 5,000 kb by pulsed field gel electrophoresis
b.direct staining--ethidium bromide intercalation:-more later
c.DNA can be recovered from gel
2.PAGE used for small fragments
a.5 – 500 bps
i.Can separate DNA's that differ by only one bp
ii.Used for sequencing gels
b.Disadvantages--Difficult to prepare and handle. Neurotoxin
B.More about agarose gel electrophoresis
1.Structure of agarose
a.Isolated from seaweed--highly purified agar
b.Repeating polymer of D-gal (14) 3,6 anhydro-L-gal. Not cross-linked
c.Can be modified so low melting temperature w/o loss of gel strength
d.Typically: 0.3% for 5-60 kb; 0.9% for 0.5-7 kb; 2% for 0.01-2 kb
2.Preparation. Hardness of gel and ease of handling depends on % gel.
a.Melt--often in microwave-- Be careful not to superheat (remember safety film?). Or melt in boiling water bath.
b.Can hold in ~ 55°C bath til ready to use
c.Pour into horizontal form, include comb to make wells. Diagram
d.After solidified, carefully remove comb after adding running buffer.
3.Electrophoresis
a.At neutral pH DNA carries – charge, (why?); migrates towards + electrode
b.Rate of migration--many factors
i.DNA size: ≈ 1/log bp for linear, double stranded molecules. Use ladders (standards) commercially available.
ii.Supercoiled DNA moves differently than linear. There are now supercoiled ladders available
iii.Nicked circles also move differently than same-sized linear pieces.
iv.Current, ionic strength, buffer choice affect migration
c.Applied voltage--too high will melt gel, distort bands. 100 V standard
i.At low voltage, linear DNA migrates ≈ voltage.
ii.As field effective size range of separation
d.Base composition and temperature don't significantly affect migration.
i. if < 0.5% agarose, or LOW melting gel, run at 4°C.
iiEthidium bromide in gel reduces mobility ~ 15%
e.Buffer considerations
i.Usually Tris (Acetate, Borate or Phosphate)
ii.~ 50 mM, pH 7.5 - 7.8 plus EDTA
4.Loading gel and visualization
a.Loading buffer:
i.Bromophenol blue (BPB) tracking dye migrates like ~ 300 bp fragment
ii.Xylene cyanol often also put in; migrates like ~ 4 kb fragment
iii.Sucrose or glycerol to make sample dense so it will sink below buffer on loading.
b.Visualization: Ethidium bromide (carcinogen) or methylene blue
i.Structure: Intercalation Fluorescence upon exposure to UV light
ii.minimum DNA ~ 2 ng/0.5 cm band with ethidium bromide.
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12b Plasmid 2
3723 F02
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12b Plasmid 2
3723 F02
c.Soak gel in EthBr (~ 0.5 µg/ml) OR include in gel (1 µg/ml).
i.free EthBr migrates towards cathode-- moves out of bottom of gel
ii.small pieces might not dye sufficiently by this method
C.Safety
1.Electrophoresis: High voltage--do not override safety features of gel boxes. Use care when turning on / off.
2.Ethidium bromide-- mutagen/carcinogen
a.Avoid powder--if must make up, gloves, mask, hood.
b.Wear gloves when handling gels
c.Disposal: protocol to inactivate (Na nitrite and hypophosphorous acid) or Column to pull it out- have Safety remove solids (EthBr contaminated) Incinerated by Safety
3.UV light--Protect eyes (UV goggles, even if wearing prescription glasses, unless they have UV coating)--if working under UV for prolonged periods of time, wear sunscreen, face shield.
D.Interpretation of gels
1.Standards: 1 kb ladder (for linear DNA only) and supercoil ladder
2.Controls: Uncut vs. cut
3.Make a record. Poleroid pictures--or scan in using Gel Doc program
E.Our purpose with gel: determine if plasmid has insert and what its size is
1.Expectations:
a.single digests--one band of total recombinant plasmid size
b.double digest --two bands
i.one at < 2.9 kb from pBluescript (w/o SacI-EcoRI fragment)
ii.Second could be any size-- is the insert
2.Determine which culture has an insert that you are interested in sequencing.
linear DNA ladders
V.DNA Concentration
A.Need to know how much DNA is in sample for sequencing reactions. Core needs ≈ 2 µg dsDNA (or ≈ 1 µg ssDNA or ≈ 0.5 µg PCR product) as sequencing template.
B.Determine from A260.
1. Nucleotides absorb UV light with maximum at 260 nm.
2.Formula: [DNA in ng/µl] = A260 x Dilution factor x 50
C.Estimate purity of DNA: DNA has A260/A280 ≈ 1.7 – 1.9; RNA has A260/A280 of ≈ 2.0 and protein has A260/A280 ≈ 0.6
D.In lab
1.While gel is running, determine concentration and amount of each DNA sample
2.After gel is done and picture taken, decide which sample you will turn in for sequencing.
VI.Problems
1.You have isolated plasmid DNA by precipitation with ethanol and dissolved it in TE buffer. To determine its concentration you add 2 µl of the DNA to 78 µl of water and read the absorbance on the DNA reader (which can read as little as 80 µl). and get the following results: A260 = 0.146; A280 = 0.082. Do you think this DNA is of a quality that can be used for sequencing, and how much of the original sample is needed to provide the Core personnel with enough DNA for sequencing?
Solution:The DNA concentration is 50. x 0.146 x 40. =292 ng/µl = 0.29 µg/µl
The A260/A280 ratio is 0.146/0.082 = 1.78 (1.8), which is correct for DNA, so the quality of the DNA is probably fine for sequencing (although it should be run out on a gel to check). Janet needs 2 µg of DNA: (0.29 µg/µl)(x µl) = 2 µg; x = 7 µl
2.If you use pBluescript II KS+ vector and genomic DNA restricted with EcoRI only in your ligation reaction, what are the possible products of the ligation (assuming no more than one molecule each of vector and genomic DNA per reaction)? What would be the result of transformation and blue/white screening of each product you made?
Solution: The three possibilities are shown below as A, B, and C.
A. regenerates an intact plasmid, which would transform well and result in blue colonies because it would have an intact -galactosidase gene.
B. gives a cyclized form of the genomic DNA fragment, which would transform into cells, but would not allow the cells to grow because no ampr gene would be present.
C. gives the recombinant plasmid, which, when transformed into DH5 cells will result in white colonies on TAXI plates because the -galactosidase gene is interrupted by genomic DNA.
3.You have isolated a recombinant plasmid prepared from EcoRI and SacI digesed vector (pBluescriptII KS+) and genomic DNA. Your restriction digestion of the recombinant plasmid does not give quite the results you expected. The SacI only digest gives one band at ~ 5 kb, while both the EcoRI only and the EcoRI + SacI digests give two bands; one at ~2.9 kb and one at ~1.9 kb. Explain these results.
Solution:Sometimes when doing double digests one or both enzymes work less efficiently than desired and some DNA molecules are cut at only one site. Apparently the recombinant plasmid you cloned here is made from a vector that was restricted only at the EcoRI site. The genomic DNA was a piece with EcoRI recognition sites at each end. When the recombinant plasmid was treated with SacI only for the restriction analysis, it was cut only at the SacI site in the MCS, giving a piece of 5 kb (total recombinant plasmid length). When cut with EcoRI only, the insert was cut out, giving pieces of ~2.9 kb (the vector) and ~1.9 kb (the insert). When cut with both enzymes (assuming both are working this time) you get the insert (1.9 kb) and two pieces from the vector (the large piece, ~2.9kb, plus the small piece of MCS between the EcoRI and SacI sites,~.05bp). However, the small piece would not likely show up on gel electrophoresis because it would bind so little ethidium bromide and it would probably have been run off the end of the gel.