Page 1 of 5 10/09 Subcloning DNA Fragments into pbluescript Preparation of pbluescript Vector 1. Digest 1 µg of pbluescript with Eco RI 2. Following digestion, add 0.1 volumes of 3M sodium acetate (ph 5.2) and 3 volumes of 95% ethanol. 3. Allow the plasmid to precipitate for at least 2 hours at -20 C or 20 minutes at -70 C. 4. Centrifuge the plasmid DNA for 15 minutes in a microcentrifuge (use the Marathon Microcentrifuge) at the top speed. 5. Resuspend the pellet in 10 µl of sterile dh 2 O. Purification of DNA fragments to be Subcloned using IBI s Gel/PCR DNA Fragment Extraction Kit 1. Using Eco RI, digest the DNA fragment you want to subclone. Digesting multiple samples and running them in different lanes of the gel is recommended because it will provide more DNA for the subcloning process. 2. Make a 1.2% (w/v) low-melting agarose gel in 1X TAE (Tris, acetate, EDTA) buffer by diluting the 50X concentrated TAE stock solution. 50X Tris-acetate EDTA buffer (for 500 ml) a. 2.0M Tris 121g b. 28.6ml of glacial acetic acid c. 50ml of 0.5M EDTA (ph 8.0) - 9.3g EDTA/50ml d. Adjust ph to 8.0 e. autoclave 3. Place the flask onto a ring stand and gently swirl the flask while heating the agarose solution over a Bunsen burner. Bring the solution to a boil and remove the flask from the burner. BE CAREFUL NOT TO LET THE AGAROSE SOLUTION BOIL OUT OF THE FLASK. 4. Allow the agarose to cool to 60 C and add 0.5 µg/ml of ethidium bromide (our stock of ethidium bromide is 10 mg/ml) and gently mix. WEAR GLOVES FROM THIS POINT ON BECAUSE ETHIDIUM BROMIDE IS A MUTAGEN! Wash the pipette tip well in water before you dispose of th tip. 5. Pour the agarose into the gel tray. Make sure the gel comb is inserted into the tray. Fill the tray with agarose until the volume reaches the top of the comb s teeth. Allow the gel to harden for approximately 20-30 minutes. 6. Add 4 µl of sterile DNA tracking dye to each sample. Mix well and centrifuge for 5 seconds. Run λ controls and add tracking dye to this sample. Prepare a sample containing a 100 bp ladder (5-6 µl) with tracking dye. Place the samples back on ice. 7. After your gel has hardened, gently remove the comb and the side supports from the gel. Place the ge into the chamber and add enough 1X TAE buffer to cover the gel.
Page 2 of 5 8. Carefully load your samples into the gel using a micropipette. Load the entire sample into the gel. Be sure to record what wells contain your samples. 9. Place the cover onto the electrophoretic chamber and connect the electrodes to the power supply. Run the gel between 50-80 volts. 10. Analyze your results under an ultraviolet light source and photograph. 11. Excise the DNA fragment(s) of interest (remove as much agarose as possible) using a clean razor blade. Transfer up to 300 mg of gel slice (from the multiple digestions) into a microcentrifuge tube. (The maximum capacity of each column is about 300 mg of gel mass.). 12. Add 500 µl of DF buffer to the sample and mix by vortexing. 13. Incubate the sample at 55-60 C for 15 minutes or until the gel slice(s) are completely dissolved. During the incubation invert the tube every 2-3 minutes. Cool the dissolved sample mixture to room temperature. (Once the agarose gel is melted, the gel will not resolidify at room temperature.) 14. Place one DF column in a 2 ml collection tube for each tube containing dissolved gel slices. 15. Transfer the dissolved gel mixture (up to 800 µl) to the DF column. 16. Centrifuge the DF column assembly in a microcentrifuge (use the larger microcentrifuge) at top speed for 30 seconds. Discard the flow-through and place the DF column back in the 2 ml collection tube. (If the samplex mixture was more than 800 µl, transfer the remaining gel mixture to the DF column and repeat step 16.) 17. Add 400 µl of W1 buffer into the DF column and microcentrifuge (top speed) for 30 seconds. Discard the flow-through.. 18. Place the DF column back into the 2 ml collection tube. Add 600 µl of Wash buffer (be sure that ethanol has been added to the Wash buffer when the kit was first opened) into the DF column and allow to incubate for 1 minute at room temperature. 19. Microcentrifuge the sample for 30 seconds (top speed) and discard the flow-through. 20. Place the DF column back into the 2 ml collection tube. Microcentrifuge (top speed) again for 3 minutes in order to dry the matrix. 21. Transfer the dried DF column to a new 1.5 ml microcentrifuge tube. Add 15-50 µl of Elution buffer into the center of the column matrix. Let the column sit for 2 minutes or until the Elution buffer is absorbed by the matrix. 22. Centrifuge (top speed) for 2 minutes to elute the purified DNA. If you started with multiple gel slices of the SAME sample that required multiple columns, combine your eluted DNA into one tube. 23. Determine the mass and concentration of your purified DNA fragment by scanning the DNA from 310-220 nm in the spectrophotometer. You will need to have a volume of 50 µl. Your peak absorbance should be between 257-260 nm.
Page 3 of 5 DNA Mass = (OD @ 257 nm) (50 µg/ml) (Volume ml). Ligation of purified DNA fragment into pbluescript 1. Add the following to a 0.5 ml PCR microcentrifuge tube: 5 µl 2X Rapid Ligation Buffer, (Vortex the 2X Rapid Ligation Buffer vigorously before each use.) 50 ng of purified pbluescript plasmid DNA PCR product (see calculation**) Use a 3:1 molar ratio of insert:vector. 3 Weiss units T4 DNA Ligase Sterile dh 2 O to a final volume of 10 µl **Calculation of PCR product mass: (ng of vector x kb size of insert) / (kb size of vector) x (insert:vector molar ratio) = ng of insert Example: You need to determine what ratio of insert to vector to run. For example, a 3:1 ratio of insert:vector (assuming the DNA fragment is 2.2 kb) is (50 ng vector)(2.2 kb insert) / (3.0 kb vector) x (3/1) = 110 ng of insert 3. Mix the reactions by pipetting. Incubate the reactions overnight at 4 C (use the refrigerated water bath). The time and temperature may have to be experimentally determined. Preparation of LB Plates with IPTG and X-Gal for Blue/White Selection 1. Inoculate 2 LB plates (containing ampicillin at 50 µg/ml) with IPTG and X-Gal per ligation reaction. 2. LB plates Bacto-tryptone Bacto-yeast NaCl Add dh 2 O to 800 ml. 10.0 g/l 5.0 g/l 10.0 g/l Adjust the ph to 7.5 Bring the volume to 1 L Add 1.5% (w/v) Bacto-Agar Autoclave and allow media to cool Add 50 µg/ml of dry ampicillin, mix well and pour about 20-25 ml into each Petri dish. 3. IPTG/X-Gal Plates a. Mix 4 µl of 200 µg/µl IPTG stock with 16 µl of X-Gal (the X-Gal stock is 50 mg/ml) per plate. b. Then add 20 µl onto the center of each LB agar plate and spread evenly (use sterile technique). c. Incubate the plates at 37 C until the fluid disappears. d. Equilibrate the plates to room temperature prior to plating.
Page 4 of 5 Transformation of the Ligation Mixture into JM109 using Blue/White Selection 1. Briefly centrifuge the samples containing the ligation reactions. Add 5 µl of each ligation reaction to a microcentrifuge tube containing 100 µl of competent JM109 cells on ice. 2. Plate 50 µl of the transformation culture onto duplicate pre-warmed LB/ampicillin/IPTG/X-Gal plates (two plates per transformation). 3. Incubate the plated cells overnight at 37 C. Approximately 20-40 colonies per plate are routinely seen. White colonies generally contain cloned insert DNA while blue colonies do not. Blue colonies should also be checked for cloned insert DNA. Plasmid Clone Isolation for Characterization using Zyppy Plasmid Miniprep Kit A. E. coli Inoculation 1. In order to determine if your transformants contain plasmids with a cloned insert, inoculate a single white colony (blue colonies do not contain inserts) from your transformation plate into sterile liquid LB (10 ml) containing 50 µg/ml of ampicillin. Inoculate up to 6 separate white colonies per plate (inoculated into 6 different tubes of LB; one colony per tube of LB). Grow the transformants overnight at 37 C in the shaking incubator (200 RPM). B. Buffer Preparations. Before beginning the protocol, make sure the following preparations have been completed. 1. Add RNase A to the Neutralization Buffer: aliquot 1 ml of Neutralization Buffer into the tube containing the lyophilized RNase A, mix and transfer the solution back into the Neutralization Buffer bottle. This buffer must be stored at 4 C. 2. Add 24 ml of 100% ethanol (or 26 ml of 95% ethanol) to the 6 ml Zyppy Wash Buffer before use. 3. The 7X Lysis Buffer may have a precipitate. If you notice a precipitate in this buffer, heat the buffer at 37 C for 15 minutes and mix the bottle by gentle inversion. C. Plasmid Isolation Protocol 1. Mix your overnight culture of cells completely and pipette 1.5 ml of cells into a microcentrifuge tube. Centrifuge the culture in the microcentrifuge for 30 seconds. Discard the supernatant. 2. Repeat step one twice using the same microcentrifuge tube. After you discard the supernatant, completely resuspend the cells in 600 µl of sterile dh 2 O. DO NOT DISCARD THE ORIGINAL OVERNIGHT CULTURE. IF THE CLONING AND TRANSFORMATION ARE SUCCESSFUL, YOU WILL NEED THIS CULTURE TO MAKE STOCKS OF THE TRANSFORMANTS. 3. Add 100 µl of 7X Lysis Buffer and mix the sample by inverting the tube 4-6 times. The solution should change from opaque to clear blue indicating complete lysis. You must proceed to the next step within 2 minutes. 4. Add 350 µl of cold Neutralization Buffer and mix thoroughly. The sample will turn yellow when the
Page 5 of 5 neutralization is complete and a yellowish precipitate will form. Invert the sample an additional 2-3 times to ensure complete neutralization. 5. Centrifuge the sample at top speed in the Marathon microcentrifuge for 4 minutes. 6. Transfer the supernatant (~900 µl) into a Zymo-Spin II column. Avoid disturbing the cell debris pellet. 7. Place the column in a collection tube (from the kit) and centrifuge for 15 seconds. 8. Discard the flow-through and place the column back into the same collection tube. 9. Add 200 µl of Endo-Wash Buffer to the column. Centrifuge for 15 seconds. Empty the collection tube. 10. Add 400 µl of Zyppy Wash Buffer to the column. Centrifuge for 30 seconds. 11. Transfer the column into a clean 1.5 ml microcentrifuge tube. Add 30 µl of Zyppy Elution Buffer directly to the column matrix and let the column stand for 1 minute at room temperature. 12. Centrifuge the column for 15 seconds to elute the plasmid DNA. V. Endonuclease Analysis of Plasmid DNA Isolated from Transformants 1. Add the proper amount of DNA (0.75 µg of λ molecular weight markers pre-digested with Hind III and/or Eco RI, 6 µl of 100 base pair ladder, 3-5 µl of isolated plasmid that you plan to digest) into separate sterile microcentrifuge tubes. Include one sample of undigested plasmid DNA as a negative control. λ Molecular Weight Markers λ Eco RI 21.2 Kb, 7.4, 5.8, 5.6, 4.9, & 3.5 λ Hind III 23.1 Kb, 9.4, 6.6, 4.4, 2.3, 2.0, 0.6, & 0.1 2. Add sterile dh 2 O so that the total volume is 18 µl. 3. Add 2 µl of the correct 10X restriction endonuclease buffer (see chart in the lab) to all plasmids that you are digesting as well as the pre-digested λ (no need to add buffer to the 100 base pair ladder or the undigested plasmid), mix well and centrifuge for 5 seconds. 4. Heat the samples you are going to digest at 65 C for 5 minutes. 5. Keep the tubes on ice. 6. Add 0.5 µl of the proper restriction endonuclease to all samples you are digesting, mix well and centrifuge for 5 seconds. 7. Incubate the samples you are digesting at the correct temperature (see chart in the lab) for at least 1 hour (preferably longer). There is no need to incubate the undigested plasmid, the λ molecular weight markers or the 100 bp ladder. 8. Heat the digested samples, undigested samples and all l molecular weight samples at 65 C for 5 minutes. Place the samples back on ice. There is no need to heat the 100 bp ladder. 9. The digested DNA can be run and analyzed on an agarose gel or frozen at -20 C until needed.