Miniprep: Plasmid purification

Transcription

1 Miniprep: Plasmid purificatin Guide fr curing plasmids frm bacteria. Three pssible kits. MN was phased ut during the igem perid. Use QIA when ding the vacuum prtcl, E.Z.N.A when using the centrifuge. QIA kit (hmemade buffers) P1 buffer must be kept n ice at all times utside the fridge. N.B.: 1 min centrifugatin at 13,000 rpm can be used instead f vacuum at each step nly use vacuum if yu have received prper instructins. Pellet 4 ml culture at > 8,000 rpm fr 3 min. Resuspend in 250 µl P1 buffer and transfer t micrcentrifuge tube. Add 250 µl P2 buffer and mix by inverting 4-6 times. Add 300 µl N3 buffer and mix by inverting 4-6 times. Centrifuge fr 5 min at 13,000 rpm. N.B.: Start cleaning spin clumn meanwhile. Transfer supernatant t new centrifuge tube and spin fr 5 min at 13,000 rpm. Discard pellet. Prepare vacuum suctin by cleaning 1x used spin clumns: Insert a 1x used spin clumn int the vacuum fr each sample. Make sure all unused valves are clsed. Apply 1 ml 0.1 M HCl t each clumn and suck them dry. N.B.: When using the vacuum pen a valve just befre turning ff the vacuum, d this as sn as the spin clumn is sucked dry. Apply 1 ml MQ H 2 3 times. The clumn shuld nw be clean and ready fr use. Apply the supernatant t the spin clumn and suck dry using vacuum. Wash by applying 0.5 ml PB buffer and sucking dry using vacuum. Wash by applying 075 ml PE buffer and sucking dry using vacuum. Transfer clumn t cllectin tube and centrifuge fr 1 min at 13,000 rpm. t remve residual washing buffer. Place spin clumn in 1.5 ml micrcentrifuge tube. T elute DNA add 50 µl EB buffer t the center f the clumn (ne drp at a time) and let it stand n the table fr 1 min. Centrifuge fr 1 min at 13,000 rpm.

2 Remve spin clumn and place in 2x used bx. DNA sample shuld nw be in tube. Stre sample at -20 C. Can be thawed and re-frzen multiple times. Buffers Generally made and stred in 50 ml falcn tubes. P1 (15 ml falcn) Must be stred at 5 C and kept n ice at all times when utside. P2 N3 PB 50 mm Tris-HCL ph 8.0 (7.88 g/l) 10 mm EDTA 100 µg/ml RNase A 200 mm NaH (8 g/l) 1% SDS (1 g/l) 4.2 M Guanidine HCl ( g/l) 0.9 M Ptassium Acetate ( g/l) Fr 50 ml: 35 ml Guanidine HCl 15 ml isprpanl PE (ph 7.5) Fr 50 ml: EB 0.5 ml 1M Tris ph ml 96% EtH 7.5 ml MQ H 2 10 mm Tris-HCl ph 8.5

3 E.Z.N.A kit Guide fr islatin f plasmid DNA frm E. cli grwn in an vernight 1-5 ml LB culture, fllwing manufacturers instructins. Get the kit, cntaining all buffers, frm the cupbard in the gel rm. Slutin I cntaining RNAse is fund in the fridge in the gel rm. Prtcl 1. Using the prtcl fr inculatins, prepare a liquid inculatin f a clny in 5 ml LB. Grw ver night befre starting this prtcl. 2. Centrifuge at 10,000 x g fr 1 minute at rm temperature. 3. Decant r aspirate and discard the culture media. 4. Add 250 μl Slutin I/RNase A. Vrtex r pipet up and dwn t mix thrughly. Cmplete resuspensin f cell pellet is vital fr btaining gd yields. Nte: RNase A must be added t Slutin I befre use. 5. Transfer suspensin int a new 1.5 ml micrcentrifuge tube. 6. Add 250 μl Slutin II. Invert and gently rtate the tube several times t btain a clear lysate. A 2-3 minute incubatin may be necessary. 7. Add 350 μl Slutin III. Immediately invert several times until a fcculent white precipitate frms. 8. Centrifuge at maximum speed ( 13,000 x g) fr 10 minutes. A cmpact white pellet will frm. Prmptly prceed t the next step. 9. Insert a HiBind DNA Mini Clumn int a 2 ml Cllectin Tube. 10. Transfer the cleared supernatant frm Step 8 by CAREFULLY aspirating it int the HiBind DNA Mini Clumn. Be careful nt t disturb the pellet and that n cellular debris is transferred t the HiBind DNA Mini Clumn.

4 11. Centrifuge at maximum speed fr 1 minute. 12. Discard the fltrate and reuse the cllectin tube. 13. Add 500 μl HB Bufer. 14. Centrifuge at maximum speed fr 1 minute. 15. Discard the fltrate and reuse cllectin tube. 16. Add 700 μl DNA Wash Bufer diluted with 100% ethanl prir t use. 17. Centrifuge at maximum speed fr 1 minute. 18. Discard the fltrate and reuse the cllectin tube. Repeat Steps fr a secnd DNA Wash Bufer wash step. 19. Centrifuge the empty HiBind DNA Mini Clumn fr 2 minutes at maximum speed t dry the clumn matrix, as residual ethanl may interfere with dwnstream applicatins. 20. Transfer the HiBind DNA Mini Clumn t a clean 1.5 ml micrcentrifuge tube. 21. Add 25 μl Elutin Buffer directly t the center f the clumn membrane. 22. Let sit at rm temperature fr 1 minute. 23. Centrifuge at maximum speed fr 1 minute. Repeat step fr a secnd elutin, yielding a higher amunt f purified plasmid. 24. Stre DNA at -20 C.

5 NucleSpin Plasmid QuickPure Guide fr curing plasmids frm bacteria. Find the kit in the gel rm. (The kit was phased ut frm September. Use E.Z.N.A kit instead) Prtcl 1 Cultivate and harvest the cells Use 1 3 ml f a saturated E.cli LB culture, pellet cells in a standard benchtp micrcentrifuge fr 30 s at 11,000 x g. Discard the supernatant and remve as much f the liquid as pssible. 11,000 x g, 30 s 2 Cell lysis Add 250 μl Buffer A1. Resuspend the cell pellet cmpletely by vrtexing r pipetting up and dwn. Make sure n cell clumps remain befre additin f Buffer A2! Attentin: Check Buffer A2 fr precipitated SDS prir t use. If a white precipitate is visible, warm the buffer fr several minutes at C until precipitate is disslved cmpletely. Mix thrughly and cl buffer dwn t rm temperature (18 25 C). Add 250 μl Buffer A2. Mix gently by inverting the tube 6 8 times. D nt vrtex t avid shearing f genmic DNA. Incubate at rm temperature fr up t 5 min r until lysate appears clear. Add 300 μl Buffer A3. Mix thrughly by inverting the tube 6 8 times until blue samples turn clrless cmpletely! D nt vrtex t avid shearing f genmic DNA! Make sure t neutralize cmpletely t precipitate all the prtein and chrmsmal DNA. LyseCntrl shuld turn cmpletely clrless withut any traces f blue. 3 Clarificatin f lysate Centrifuge fr 5 min at 11,000 x g at rm temperature. 4 Bind DNA Place a NucleSpin Plasmid QuickPure Clumn in a Cllectin Tube (2 ml) and decant the supernatant frm step 3 r pipette a maximum f 750 μl f the supernatant nt the clumn. Centrifuge fr 1 min at 11,000 x g. Discard flw-thrugh and place the NucleSpin Plasmid QuickPure Clumn back int the cllectin tube. Repeat this step t lad the remaining lysate.

6 5 Wash silica membrane Add 450 μl Buffer AQ (supplemented with ethanl). Centrifuge fr 3 min at 11,000 x g. Very carefully discard the cllectin tube and the flw-thrugh and make sure the spin cup utlet des nt tuch the wash buffer surface. therwise repeat the centrifugatin step. 6 Dry silica membrane The drying f the NucleSpin Plasmid QuickPure Clumn is perfrmed by the 3 min centrifugatin in step 5. 7 Elute DNA Place the NucleSpin Plasmid QuickPure Clumn in a 1.5 ml micrcentrifuge tube (nt prvided) and add 50 μl Buffer AE. Incubate fr 1 min at rm temperature. Centrifuge fr 1 min at 11,000 x g.