Purification of DNA Oligonucleotides

Transcription

1 Purification of DNA Oligonucleotides Polyacrylamide gel: In a beaker, add 30 ml 30% acrylamide solution (To make this solution, add 29 g acrylamide to 1 g bis-acrylamide, bring to 100 ml with dh 2 O) 31.5 g urea (to denature) 7.5 ml 10x TBE 13.5 ml H 2 0 Heat on low and stir to dissolve urea Filter the solution (use vacuum pump in culture room, 0.45 micron pore size) Set up rig Clean plates with soap if really dirty & dry Wash with ethanol Spray small plate with Acrylease Rinse off with deionized water Buff dry When rig is set up and ready for gel, add to the filtered solution 0.5 ml 10% ammonium persulfate (10% = 50 mg/500 μl, for example) 35 µl TEMED (this makes it polymerize) Mix thoroughly in flask and pour into rig using 25 ml pipette. Let it set for about 30 minutes Pre-run gel for 30 min at 400 V (tank buffer: TBE 1X) Resuspend samples: Turn water bath to 90 C. Make TE in a 50 ml conical tube: 500 µl 1 M Tris, ph µl 0.5 M EDTA Adjust to 50 ml with dh 2 0 Add 50 µl TE to each sample and let sit for 2 min. (The scale we order is 50nmol) Vortex 30 sec. Flash spin sample down to bottom of tube. Add 25 µl sample to eppendorf tube (FREEZE ORIGINAL TUBES) 25 µl formamide 1 µl bromophenol blue - 1% stock solution Heat mixture 3 min at 90 o C. Run Electrophoresis: Rinse wells of gel using tank buffer and syringe to get the urea out (they should be filled with buffer) Load wells with resuspended samples

2 Run rig at 400 V in 1 X TBE for 2-3 hours until blue is ¾ of the way down the gel. Post-Electrophoresis Treatment: Remove plate treated with Acrylease and leave the gel stuck to the other plate Place gel (still on plate) in a pyrex dish containing a mixed solution of 300 ml water 60 µl ethidium bromide (10 mg/ml) Place pyrex on table top shaker 5 min at speed 3 Gel Excision: Find DNA on UV box (will look like a dark blob) Cut out DNA blob with a scalpel and put into a 14 ml falcon tube to transport. Elution of Oligos from Polyacrylamide Gel: Crush gel in parafilm and funnel it back to falcon tube with 5 ml TE. Incubate overnight at 37 o C in shaker Next Day Filter eluate using 5 ml syringes Syringe filters Collect in 15 ml conical tubes put on filter, take out plunger, pour in solution and replace plunger. Concentrate eluate by successive extractions in 1 vol. sec-butanol by: Add sec-butanol (1 ml per ml of solution) A) Vortex well B) Centrifuge 2 min (use the one that is in the cold room doesn t necessarily have to be cold) at rpm C) Remove top layer (sec-butanol) with pipette and discard Repeat steps a through c using equal volumes of sec-butanol and DNA solution Repeat steps a & b; remove DNA solution (bottom layer) and put it into an eppendorf tube. Final volume should be less than 500 µl. (Don=t depress pipette all the way so you can expel the little amount of sec-butanol that enters the tip as the pipette tip travels through the secbutanol to your sample at the bottom.) Place waste sec-butanol in an organic waste bottle Add TE up to the 0.5 ml mark of eppendorf tube. Then Add 33 µl NaCl (0.2 M) - 3 M stock Add 5 µl MgCl 2 (10 mm) - 1 M stock Add 1,250 µl EtOH Close tubes, mix (vortex about 2 min) and put in -20 o C freezer overnight or -80 o C for 15 min.

3 Spinning: Centrifuge 15 min at 14,000 rpm in cold room Take off top layer (pellet should be clearly visible) Wash pellet by adding approximately 1 ml of cold 80% EtOH Centrifuge 15 min at 14,000 rpm in cold room Remove liquid Flash spin Remove liquid with a 10 µl pipette Let pellet dry (cover with a kimwipe it s very easy to loose!) Resuspend oligos in 30 µl TE (Depends on the size of your pellet, stay between µl) Put in 4 o C refrigerator for overnight, or move ahead to the next step. Measure O.D. of ssdna: Vortex tubes 30 sec In a separate set of eppendorf tubes, add: 2 µl sample 498 µl dh 2 O Measure O.D. using dh 2 O as the reference sample Compute: OD 260 x 30 (1 OD = 30 µg/ml) x 250 (dilution) = µg/ml ssdna Anneal the Complementary Strands: Make TNE by adding into a larger falcon tube: 500 µl 1M Tris ph µl 0.5M EDTA 1.67 ml 3M NaCl Adjust total to 50 ml with H 2 0 In your original set of tubes (the ones from which you extracted the 2 µl of sample), add TNE so that all tubes are 1 µg/1 µl Example 3,570 µg/ml = ssdna 3,570 µg = x µg left in your tube after taking out 2 µl 1000 µl 28 µl (if added 30 µl) x = 100 µg (this is what is present in your tube) You want 100 µg 100 µl you must add 72 µl of TNE to your volume of 28 µl already in the tube to come out with 1 µg/1 µl Add your two complementary strands together in a 1:1 volume Example (1A) 3,570 µg = x, x = 100 µg + 72 µl TNE = 100 µl 1000 µl 28 µl (1B) 1,403 µg = x, x = 39 µg + 11 µl TNE = 39 µl 1000 µl 28 µl

4 Add in another eppendorf tube 39 µl of each sample (1A and 1B) - 39 µl is your limiting volume. You now have half the number of eppendorf tubes and you must boil them for 10 min: Boil water in a beaker Place eppendorf tubes in a holder that will hold them in the water with their caps out of the water for 10 min. Remove beaker from hot plate (tube holder with tubes still in beaker), cover loosely with foil and let cool 3 hours at room temperature Place in 4 o C refrigerator (if storing) NOTE: This is now double stranded DNA Measure OD of dsdna: In a separate eppendorf tube, add: 5 µl sample 495 µl water Measure OD using water as a reference sample Compute for dsdna: OD 260 x 50 (1 OD = 50 µg/ml) x 100 (dilution) = µg/ml dsdna Phosphorylation of Double-Stranded Oligos: Calculate volume of double stranded oligo needed for 5µg Example: 4612 µg/ml dsdna 4612 µg = 5 µg x = 1.08 µl 1000 µl X 2) Mix: 5 µg primer (template) 4 µl buffer (10X T4 PNK (polynucleotide kinase buffer) in freezer) 4 µl ATP 10mM (in freezer) 1 µl T4 kinase - add last (in freezer) Enough water to bring total volume of mixture to 40 µl 3) Incubate 30 min at 37 o C in waterbath 4) Add 40 µl phenol:chloroform:isoamyl (25:24:1) shake bottle (in big fridge, in foil) a) vortex b) centrifuge 5 min c) Keep upper phase (suck out lower phase put in an epptube) 5) Add 40 µl chloroform:isoamyl (24:1) shake bottle (in big fridge, in foil) Vortex centrifuge 5 min Keep upper phase (suck out lower phase and put in an epptube discharge in waste bin halogenated- organic)

5 6) Precipitate by adding 0.5 µl 1 M MgCl 2 (final conc: 10mM) 3.3 µl 3 M NaCl (final conc: 200mM) 100 µl EtOH (100%) Keep 1 hour at -20 o C or 15 min at - 80 o C Centrifuge 15 min in cold box - remove supernatant (centrifuge in fridge) Wash pellet with 70% EtOH (centrifuge 5 min) ~ 1ml (cold) a) Take off EtOH b) Centrifuge and remove last drops c) Dry pellet d) Resuspend in 25µl TE (= 200ng/µl) e) Leave in 2 mins and vortex (can be put in freezer -20 C) Digestion of Bluescript 2 µg DNA 2 µl 10x green FD (fast digest) buffer B 1 µl FD BamH1 1 µl FD BglII Adjust total volume to 20 µl using ddh20 Digest 20 min at 37 C in water bath, then inactivate for 5 min at 80 C Add 2 µl CIAP (calf intestine alkyline phosphate) And 2.5 µl CIAP 10x buffer to dephosphorylate Incubate 30 min at 37 C inactivate at 75 C for 15 minutes. (waterbath) Run gel (Low melt) Cut out (to purify insert from vector) and do ligation: Visualize digestion products on Agarose gel Make low melt agarose. (0.5 g l.m. agarose in 50 ml 1x TAE for a small gel) Heat up and dissolve powder, after cooling down a bit, add 2.5 μl ethidium bromide. Poor gel in gel chamber, put in comb. Let it polymerize for 45 min 1 hour. Make up 280 ml 1x TAE buffer and add 12.5 μl ethidium bromide. (must use TAE for low melt) Use 6μl DNA ladder mix + 4μl dh 2 O Load gel. Let it run at 90 V for about 45 min- 1 hour. Picture of the gel Go to Canon Zoom browser, choose your folder edit remote capture view finder off. Put the gel inside (wells facing you) turn on the regular light and make sure that size/quality is on, flash off and macro off and that white balance is fluorescence. Then zoom in and choose the best size. Turn off the light, on the UV (green light in the front) then release capture. Print the image by right clicking on the mouse and print. Choose continue and then a new image comes up, right click again and choose print. Bluescript is about 3kbp. Heat digested vector at 65 C in waterbath for use in ligation.

6 Ligation: 1) Calculate ng insert needed by: ng vec x kb insert x molar ratio (usually 3:1, but in this case, 40:1) insert = ng insert kb vector vector From phosphorylation, left with 200 ng/µl 200 ng = ng 1 µl x volume of insert needed Mix: x µl (200 ng) vector p95bs, cut with Bam HI and Bgl II (see above) x µl (200 ng) insert µl H 2 O (adjust so whole mix totals 10 µl) 1 µl 10 x ligase buffer 1 µl ligase (T4 DNA ligase) 10 µl Mix by flicking tube and then flash centrifuge to collect in bottom of tube Incubate at 16 o C overnight. Illustration of the digestion: BglII HindIII Bam HI Hind III Bgl II Bam HI Hind III

7 Transformation: *Make IPTG/X-gal plates. White=insterted, blue=not. 1. Thaw 100 μl (per sample) of competent cells gently warm between hands. 2. Label 5 ml Falcon tubes. 3. Add 100 μl of cells into each Falcon tube. Keep on ice for 10 minutes. 4. Add 5 μl ligated DNA to Falcon tubes. a. Swirl tubes gently. b. Place on ice for minutes. 5. Heat shock cells. a. Place tubes in 42 C water bath for 45 seconds. b. Rapidly transfer tubes to ice for 1-2 minutes. 6. Add 900 μl LB medium. a. Transfer to shaking incubator at 37 C for 30 minutes. Warm LB/amp plates in incubator at this time as well. (can use X-gal plates) 7. Plate 500 μl of transformation on LB/amp plates. a. Put some sterile beads on plate b. Add 500 μl of transformation c. Gently spread cells d. Leave plates at room temperature for a few minutes until liquid is absorbed e. Dump off glass beads into 100% EtOH 8. Invert plates and incubate at 37 C for hours (no more than 20 hrs). Picking Colonies: 1. Label 14 ml Falcon tubes 2. Add 5 ml LB medium and 5 μl 1000X ampicillin (50 mg/ml) 3. Pick one colony for each tube 4. Put in shaking 37 C O/N Next Day: 1. Vortex tubes to mix bacteria in case they have settled on the bottom 2. Pour bacteria into eppendorf tubes until the tubes are full 3. Centrifuge for 1 min. (14,000rpm) and pour off supernatant. Bacteria is ready to miniprep. Do Sigma Spin Miniprep Kit Protocol: After miniprep, send μl miniprepped DNA in Eppendorf tube with 5 µl of primer to thedna sequencing center. Fill out submission form on BYU DNA Sequencing Center s web site.

8 Double Digest: Add the following to a 1.5 ml Eppendorf tube: (two different tubes) Vector 17 μl DNA from miniprep 2 μl 10X Buffer B if using Fisher brand 0.5 μl BglII 0.5 μl HindIII Insert 17 μl DNA from miniprep 2 μl 10X Buffer B if using Fisher brand 0.5 μl BamHI 0.5 μl HindIII Digest 20 μl total mixtures at 37 C for 2 hours. Run digest on 1% agarose gel (see above) and QIAEX. Ligation: 2 μl vector 6 μl insert 1 μl 10X ligase buffer 1 μl ligase Incubate 10 μl total at 16 overnight. Transform ligated DNA and pick colonies (see above). Then repeat digestion/gel/qiaex/ligation/transformation/miniprep process until you have a 4x insert.