Experiment 3: Plasmid Digestion, Ligation, E coli. Transformation, and Selection (CSS451, 2010) GENERAL COMMENTS 1. It is the accepted convention that 1 unit of restriction endonuclease corresponds to the amount of enzyme required to completely digest 1 ug of lambda DNA in 1 hr under optimal assay conditions. 2. Activity of restriction endonuclease can be affected by: a. The ph of the reaction solution, b. The presencee of divalent cations in the reaction solution, c. The presencee of organic solvents in the reaction solution, d. The concentration of glycerol in the reaction solution (it should be no greater than 5% in the final mixture), e. The ionic strength of the reaction solution, f. The mean base composition of the nucleotides adjacent to the recognition site. 3. Repeated freezing and thawing of the DNA sample will damage the material 4. VERY IMPORTANT!! Restriction endonucleases must be stored at -20oC. They should not be left at room temperature or on ice for long periods of time. 5. Use presterilized plastic eppendorf tubes and plastic pipette tips to provide sterile conditions. 6. Mixing several restriction endonucleases in a single reaction solution may lead to the inhibition of the reaction. 1
DIGESTION: 1. Place a sterile eppendorf tube on ice. In order, add the following: Reaction system Hind III Hind III + Xba I Hind III + Xba I Xba I Sterile water 15.9 µl 15.8 µl 15.8 µl 10x high salt buffer 2 ul (NEBuffer 2, 100%) 2 ul (NEBuffer 2, 100%) 2 ul (NEBuffer 2, 100%) 10x BSA -- --- --- 2 ul Plasmid or other 2 µl (1 µg), 2 µl (1 µg), 2 µl (1 µg), bar DNA sample psk+ psk+ Restriction endonuclease (10 u/ul)* 0.1 µl 0.1 µl Hind III 0.1 µl Xba I 0.1 µl Hind III 0.1 µl Xba I 2 ul (NEBuffer 4, 100%) 20 ul 20 µl 20 µl 20 µl * One unit is defined as the amount of enzyme required to digest 1 µg of λ DNA (dam - /Hind III digest) in 1 hour at 37C in a total reaction volume of 50 µl. a. Sterile water b. 10x high salt buffer c. 10x BSA d. DNA sample e. Restriction endonuclease (10 u/ul) Xba I Activity in NEBuffers: Recognition Site: Hind III Activity in NEBuffers: Recognition Site: NEBuffer 1: 0% NEBuffer 2: 100% NEBuffer 3: 75% NEBuffer 4: 100% NEBuffer 1: 50% NEBuffer 2:100% NEBuffer 3: 10% NEBuffer 4: 50% 2. The amount of each component to be added to the reaction solution is based on the amount of DNA to be digested. The concentration of the DNA in the sample should be based upon fluorometric or spectrophotometric analysis. For the plant DNA digestions, 20 ug of DNA must be digested per reaction. For plasmid DNA, 200 ng of DNA is needed for each digestion. As an example, if your DNA sample concentration was 1 ug/ul and you want to digest 4 ug of DNA, and your total reaction mixture volume is to be 20 ul**, then add sequentially: a. 11 ul of sterile water b. 2 ul of high salt buffer c. 2 ul of 10x BSA d. 4 ul of DNA 2
e. 1 ul of restriction endonuclease **Note: Try to keep the digestion reaction to a maximum of 25 ul. (A larger volume will be needed for a 20 ug digest.) 3. Centrifuge the eppendorf tubes in the microfuge with a 1-2 second burst to collect the reaction mixture in the bottom of the tubes. 4. Place the eppendorf tubes in a 37ºC water bath or incubator for 2 hr or overnight. The use of Styrofoam boats or another floating apparatus keeps the tubes upright in the water. 5. After the digestion is completed, add the endonuclease reaction stop mix to the reaction solution or place at -20ºC. The stock solution is considered a 10x concentration, therefore add if you have a 20 ul volume reaction then addd 2 ul of the stop mix. DEL EXTRACTION and PURIFICATION of DIGESTED PLASMID (or FRAGMENT): 1. Run agarose gel. 2. Recover the target fragments from gel. 3. Quantitate DNA concentration of the recovered fragments. DPHOPHORYLATING DNA with Calf ntestinal alkaline phosphatase (CIP) 1. Suspend DNA in 1X NEBuffer (0.5 μg/10 μl). 2. Add 0. 5 unit/μg vector DNA. 3. Incubate 60 minutes at 37 C. 4. Purify DNA by gel purification, spin-columnn purification or phenol extraction. LIGATION: Calculating Insert Amount 3
****The total DNA concentration in a ligation reaction should be between 1-10 ng/ul (according to NEB). ****The insert to vector molar ratio can have a significant effect on the outcome of a ligation and subsequent transformation step. Molar ratios can vary from a 1:1 insert to vector molar ratio to 10:1. It may be necessary to try several ratios in parallel for best results. 10μL Ligation Mix Larger ligation mixes are also commonly used 1.0 μl 10X T4 ligase buffer 8.5 μl mixture of [vector and insert volume + ddh 2 O--- 3:1 molar ratio of insert to vector (~10-100 ng vector)] 0.5 μl T4 Ligase 10 μl 1. Add appropriate amount of deionized H 2 O to sterile 1,5 ml tube 2. Add 1 μl ligation buffer to the tube. Vortex buffer before pipetting to ensure that it is well-mixed. Remember that the buffer contains ATP so repeated freeze, thaw cycles can degrade the ATP thereby decreasing the efficiency of ligation. 3. Add appropriate amount of insert to the tube. 4. Add appropriate amount of vector to the tube. 5. Add 1μL ligase. Vortex ligase before pipetting to ensure that it is well-mixed. Also, the ligase, like most enzymes, is in some percentage of glycerol which tends to stick to the sides of your tip. To ensure you add only 0.5 μl, just touch your tip to the surface of the liquid when pipetting. 6. Let the 10 μl solution sit at room temperature (22-25 C) for 30 mins 7. Store at -20 C or start E. coli transformation. E. Coli TRASFORMATION 1. Thaw your competent cells on ice for 10 min. 2. Add your DNA to the cells. Don't add too much, for transforming plasmid DNA 10 picograms is enough! In case of a ligation transform half of your ligation or less. Always keep in mind that for transformation less is more! 3. Leave your cells + DNA on ice for another 10 min. 4. Put the eppendorf from the ice straight to 42C for 45 seconds (or 90 sec). 5. Place on ice for 2 min. 4
6. Add 500 microliter SOC or LB (no Aantibiotic!) to your transformed cells, transfer everything 7. Put on a shaker at 37C for 45min to 1 hour. 8. Plate out 100 microliter on a LB-plate + antibiotic + X-gal/IPTG) IPTG and X-gal for blue/white selection Stock Solutions IPTG Isopropyl thiogalactoside, or isopropyl beta-d-thiogalactopyranoside. 0.1 M solution. The formula weight is 238.3, so this is 0.238 g in 10 ml of water. Sterilize by filtration, then store in the freezer. X-gal 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside. 20 mg/ml solution. It must be dissolved in DMSO (dimethyl sulfoxide) or dimethyl formamide, not water! It must be wrapped in foil to protect it from the light, sterilize by filtration, and then stored in the freezer. Using IPTG and X-gal for blue/white selection on Petri plates There are three basic methods: spread the chemicals on top of the plates before you use them, pour the plates with IPTG and X-gal in them, or incorporate the chemicals into top agar. Putting IPTG and X-gal on top of pre-made agar plates. Spread 40 ul of IPTG and 40 ul of X- gal on top of the plate with a hockey stick spreader. Then, let the plates dry before you use them. This should take 30 minutes or so if the plate is dry (i.e. a day or two old), but up to several hours for freshly made plates. Incorporating IPTG and X-gal into the plates before pouring. After autoclaving the media and cooling it to 65oC or less, add IPTG to a final concentration of 0.1 mm IPTG ( 1 ul IPTG stock solution per ml of media) and X-gal to a final concentration of 40 ug/ml (2 ul of X-gal stock solution per ml of media). Also be sure to add the selection antibiotics at this time: usually ampicillin to a final concentration of 100 ug/ml. Putting IPTG and X-gal into top agar. This method is generally used for bacteriophage, but also works for bacterial colonies. Use 3 ml of 0.7% agar (or agarose if you want DNA that can be cut with restriction enzymes) kept at 50oC. Add 10 ul IPTG stock and 40 ul of X-gal stock. Then add the bacteria and phage mixture, mix quickly by rolling the tube between your palms, and pour it onto the plate. 5
NOTES: Xba I Recognition Site: Source: A E. coli strain that carries the XbaI gene from Xanthomonas badrii (ATCC 11672). Reagents Supplied: NEBuffer 4 (10X) BSA (100X) Enzyme Properties Xba I Activity in NEBuffers: NEBuffer 1: 0% NEBuffer 2: 100% NEBuffer 3: 75% NEBuffer 4: 100% When using a buffer other than the optimal (supplied) NEBuffer, it may be necessary to add more enzyme to achieve complete digestion. Methylation Sensitivity: dam methylation: Blocked by overlapping dcm methylation: Not sensitive CpG methylation: Not sensitive More information about: Methylation Sensitivity Heat Inactivation: 65 C for 20 minutes Survival in a Reaction: (+ + +) Suitable for an extended or overnight digestion. Enzyme is active > 8 hours. More information about: Extended Digests with Restriction Enzymes 6
Reaction & Storage Conditions Reaction Conditions: 1X NEBuffer 4 Supplemented with 100 μg/ml Bovine Serum Albumin Incubate at 37 C. 1X NEBuffer 4: 20 mm Tris-acetate 50 mm potassium acetate 10 mm Magnesium Acetate 1 mm Dithiothreitol ph 7.9 @ 25 C Unit Definition: One unit is defined as the amount of enzyme required to digest 1 µg of λ DNA (dam - /HindIII digest) in 1 hour at 37 C in a total reaction volume of 50 µl. Concentration: 20,000 units/ml and 100,000 units/ml Unit Assay Substrate: λ DNA (dam - /Hind III digest) Storage Conditions: 10 mm Tris-HCl 50 mm NaCl 1 mm Dithiothreitol 0.1 mm EDTA 200 µg/ml BSA 50% Glycerol ph 7.4 @ 25 C Storage Temperature: -20 C Diluent Compatibility: Diluent A 7