质粒DNA提取、酶切及琼脂糖凝胶电泳

Plasmid DNA Preparation for Recombination Lin Chengyu Bio 04 2010030007 ; Cooperator: Yuan Xiaowen Experiment Date: 2012-03-15; Submit Date: 2012-03-181Introduction1.1Background InformationPlasmid is a small, independently replicating and cytoplasmic DNA molecule that canbe transferred from one organism to another. It is a kind of double-stranded and usuallycircular DNA, whose size range from 1 kb to 200 kb.Figure 1 Sketch of plasmid nucleoid and cellBecause of its property, plasmid is often used as cloning vector carrying foreign genesinto the host cells. In order to make plasmid ready for recombination, severalpreparative processes shall be done, including plasmid DNA extraction, restrictionenzyme digestion and agarose gel electrophoresis.1.2Objectives(1)Learn the characteristics of plasmid.(2)Purify the plasmid DNA by alkaline lysis method.(3)Measure the DNA concentration by spectrophotometer.(4)Learn the characteristics of restriction endonuclease, and use Eco R I and Xho I toperform restriction enzyme digestion.(5)Perform agarose gel electrophoresis to separate DNAs, and compare the differenceamong original plasmids, single digestion, and double digestion.1.3Major principles1.3.1Plasmid DNA extraction(1)Some special elements, including heat, extreme pH, organic solution candenature DNA and will bring DNA into sediment;(2)When denaturing conditions are eliminated, DNA will be renatured, and itsspeed will depend on the size and ratio over protein;(3)The isolation kit has special material on the column and can specificallybind with DNA molecules. Thus, plasmids can be purified;(4)Double-strand DNA has optical density at wavelength 260nm. Itsconcentration can be measured by spectrophotometer.1.3.2Restriction enzyme digestion(1)Type II restriction endonuclease can recognize specific nucleic acidsequence and incise double strain DNA in specific sites in this sequence.Figure 2 illustrate the specific sequence Eco R I recognize and its cuttingsite;Figure 2 Cutting site of Eco R I(2)Most plasmids have multiple cloning sites (MSC) which is artificiallydesign for genetic engineering (See Figure 3). Restriction endonuclease candigest in the specific site, produce sticky ends for recombination.Figure 3 Plasmid profile (pCMV - Myc)1.3.3Agarose gel electrophoresisIn order to confirm that the preparation above is successful, agarose gel electrophoresis is essential.Agarose gel has small pores in it, whose size depends on agarose concentration. In the electric field and buffer in neutral pH, negatively charged nucleic acid will migrate toward the positive pole. Molecular weight of DNAand DNA conformation will be the major factors that determine the mobilityspeed of DNA. Thus, DNA of different sizes and conformations can beseparated. If a DNA maker exists, the size of sample can be known as well. (SeeFigure 4)Figure 4 Agarose gel electrophoresis:(1) plasmids (2) Eco R I digestion (3) double digestion12Experiment Operation2.1MaterialE.coli DH5 harboring pCMV-Myc-T10 (SIPAR),Eco R I and Xho I restriction endonuclease (in glycerol, for details see Table 1),Table 1 Recognition Sequence and cutting site of Eco R I and Xho I2NEB 1kb DNA Ladder (for details see Figure 5);Figure 5 NEB 1kb DNA Ladder32.2Chemicals and apparatus2.2.1SolutionsTable 2 Solutions2.2.2Apparatus(1)Pipettes;(2)Eppendorf tube;(3)Biodev plasmid isolation kit;(4)Centrifuge;(5)Spectrophotometer;(6)Electrophoresis apparatus;(7)UV gel imaging system.2.3Procedure2.3.1Plasmid extraction and purification(1)Pipette 1.4 ml LB medium containing E.coli into one Eppendorf tube,centrifuge at 12,000 rpm for 2 min, and discard the supernatant. Thenpipette another 1.4 ml, and repeat;(2)Add 100 µl Solution I (contains DNase-free RNase), resuspend pelletthoroughly. V ortex might be necessary. Incubate for 5 min at R.T.;Notes: Solution I provides a buffer system for materials in E.coli,avoiding harsh environment.(3)Add 150 µl Solution II, mix by inverting the tube 4-6 times gently. Solutionin the Eppendorf tube becomes clear. Incubate the tube on ice for 2 min;Notes: Solution II contains NaOH and SDS, which provide a stronglydenaturing environment. It will lyse the cell and release genomic DNA,plasmid DNA and protein into the solution. DNA is denatured and itssolubility in water is lowered.(4)Add 150 µl Solution III, mix by inverting the tube gently several times.White pellet is formed as Solution III is added. Incubate the tube at R.T. for5 min to get genomic DNA precipitate completely, and centrifuge at 12,000rpm for 8 min;Notes: Solution III can neutralize the denaturing environment caused bySolution II, so that plasmid DNA molecules renature. Sodium ion in SDSwill be replaced with potassium ion in Solution III, and becomes PDS(Potassium Dodecyl Sulfate), which is insoluble in water. PDS,combined with protein and genomic DNA, will coprecipitate into pellet.(For details, see discussion session.) Using centrifuge can removegenomic DNA;(5)Add 420 µl Binding buffer to the mini-spin column to activate its ability tobind DNA molecules;(6)Transfer the supernatant from Step 4 to the mini-spin column, mix withbinding buffer by pipette, place the mini-spin column in the collection tube,and centrifuge at 12,000 rpm for 30 sec. Discard the waste solution in thecollection tube;Notes: At this time, plasmids specifically bind to the material in themini-spin column;(7)Add 700 µl Wash buffer (contains 3 folds volume of absolute ethanol) tothe mini-spin column, and centrifuge at 12,000 rpm for 1min. Discard thewaste solution in the collection tube. Then repeat with another 500 µl;Notes: Wash buffer can wash off the non-DNA substance thatnon-specifically binds to the mini-spin column. Ethanol is essentialbecause plasmids will be washed off with its absence.(8)Centrifuge the mini-spin column as well as collection tube at 12,000 rpmfor 2 min to get rid of ethanol;Notes: Ethanol is harmful for most enzymes. Its existence will greatlyinfluence the following digesting step. Step 8 is designed for eliminateethanol from the column.(9)Replace the collection tube with a new Eppendorf tube, add 50 µl ddH2O tothe mini-spin column, incubate 5 min at R.T., and centrifuge at 12,000 rpmfor 1 min;(10)L abel the Eppendorf tube containing plasmids at initial concentration withclass, name, group on it;(11)P ipette 96 µl ddH2O into a new Eppendorf tube, and add 4 µl of plasmidsolution. Measure its concentration by spectrophotometer.Notes: [dsDNA] = 50 * (OD260– OD310) * dilution factor.2.3.2Restriction endonuclease digestion(1)Label 3 Eppendorf tube as No.1, 2, 3;(2) Add substrate, solutions, and enzymes as shown in Table 3;Table 3 Scheme of restriction endonuclease digestion(3) Incubate 3 tubes at 37 ℃ for 30 min; 2.3.3Agarose gel electrophoresis(1) During enzyme digestion, place a clean electrophoresis mold on ahorizontal surface, carefully insert a comb (15 µl slots) into the mold;(2) Pour the agarose gel solution (contains 0.8 g agarose in 100 ml TAE) intothe mold gently to avoid bubbles. The height of gel shall be slightly higherthan the black line on the side face;(3) Wait for approximately 30 min for solidification, when enzymatic reactionscomplete as well;(4) Add 10 µl 3×Loading buffer to each Eppendorf tube, and mix with pipette;(5) Carefully remove the comb, place the mold in the electrophoresis chamber,and add 1×TAE buffer in order to cover the gel to a depth ofapproximately 1 mm;(6) Load 15 µl from each Eppendorf tube into the slots of the gel: one slot forone tube. Load 4 µl of 1kb DNA ladder into the slot beside the sample;Notes : this step shall be done as quickly as possible. If electrophoresisstarts late after samples loading, samples might diffuse in the gel and causethe band get in the end not parallel with the slot.(7) Start electrophoresis immediately samples loading at 100 V, and stop whenBromophenol blue is 2/3 gel length from the starting line;(8) Place the gel into EB working solution for 20 min, and observe under UVat 310 nm. The red fluorescent bands show where DNA is and theirdarkness show the quantity. Then take photograph for records;3 Result & Discussion3.1 Plasmids extractionTable 4 Plasmid concentration and purityTable 4 shows that the plasmid extracted was 188.4 ng / µl, but not pure enough. OD 260 / OD 280 shall be 1.8~2.2. The major reason might be that during transferring supernatant from the Eppendorf tube to the mini-spin column, the pellet was loosely attached, causing some protein solute, and making the extraction not pure enough.Q1: During plasmid extraction, when Solution II was added, why can’t incubate longerthan 2 min?A1: NaOH can produce a strong alkaline environment to lyse cells, but genomic DNA will also degraded slowly under alkaline situation.Q2: When Solution III is added, white pellet can be see immediately. What is it?A2: In order to study the nature of the pellet, I designed an experiment and perform it in the lab.Table 5 Experiment designed to study the pellet after Solution III addedFrom the result, we can know that NaOH is mainly used for lyse, and denatured DNA is still soluble! Pellet produced in experiment No.2 is mostly protein that precipitate under acidic and high salt concentration environment.The pellet we see in experiment No.1 is actually mainly caused by PDS, as demonstrated among experiment 4, 5, and 6. As a result, PDS precipitate in a large amount, and its hydrophobic group binds to protein and makes it coprecipitate. Genomic DNA molecules are bind with proteins, and are brought into pellet in the same time. 3.2Gel electrophoresisThe result is shown in Figure 6.Figure 6 Plasmid DNA gel electrophoresis:Lane I – Plasmid without treatment;Lane II –Eco R I single digestion;Lane III –Eco R I and Xho I double digestion;Lane M – 1 kb DNA LadderAs shown in Figure 6, Lane III, which contains plasmids treated by double digestion,has bands at approx. 1.8 kb and 4.0 kb, and add up to approx. 5.8 kb; Lane II, whichcontains plasmids treated by Eco R I single digestion, has band at approx. 6.0 kb, whichis about the same length as the sum of two composition from Lane III.This phenomenon suggests that in pCMV-Myc-T10, the number of Eco R I recognitionsites and Xho I are the same. According to the plasmid profile, both enzyme have andonly have one recognition site, which confirm our conjecture.As for Lane I, there is no apparent band as shown in other groups. It is mainly becauseof a loading mistake. When we were loading the first sample, we failed to put the pipettenear the bottom of the slot, but the middle of it, causing a lot of DNA moleculesoverflow from the slot.4ConclusionThe plasmid sample extracted from E.coli is 188.4 ng / µl. And it is a circular DNA molecule composed of approx. 6 kilobasepairs, has 1 Eco R I and 1 Xho I restriction enzyme recognition site.5Reference【1】Results of student experiment, 2004-03-15【2】/rebase/rebase.html【3】/nebecomm/products/productN3232.asp6Appendix6.1Difference between plasmid DNA and genomic DNA extractionPlasmid DNA extraction procedure includes denaturing and renaturing, in order toremove genomic DNA; genomic DNA extraction tends to focus more on proteinremoval, and will inevitably mix with plasmid DNA if without further purification.6.2Restriction MapThe gene sequence comes from NCBI, which coding for PDCD11 (Programmed CellDeath), also named as ALG-4 (Apoptosis-Linked Gene). It is composed of 6000 bp.Using Bioedit to analyze:Use “Sequence -> Select positions” to select the whole sequence;Use “Sequence -> Nucleic acid -> Restriction map”:Figure 7-1Click “Generate Map”, here shows analysis from 881 to 960 bp:Figure 7-2And those enzymes that have no recognition sites are listed as follows:Figure 7-3。