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<b> <h3> 1. Miniprep (using Omega protocol)</b> </h3> | <b> <h3> 1. Miniprep (using Omega protocol)</b> </h3> | ||
− | <DT> | + | <DT><span>1.1 Grow 1-5mL culture overnight in a 10mL-20mL culture tube.</span> |
− | <DT> | + | <DT><span>1.2 Centrifuge at 2500xg for 5 minutes at room temperature. Decant or aspirate and discard the culture media.</span> (Original protocol called for 10,000xg for 1 minute, but the speed and time above seemed to produce better results.) |
<DD>1.2.1 Original protocol called for 10,000xg for 1 minute, but the speed and time above seemed to produce better results.<br> | <DD>1.2.1 Original protocol called for 10,000xg for 1 minute, but the speed and time above seemed to produce better results.<br> | ||
− | <DT> | + | <DT><span>1.3 Add 250uL of Solution I mixed with RNase A (pre-added). Vortex to mix thoroughly. Transfer the suspension into a new 1.5mL microcentrifuge tube.</span> |
− | <br><DT> | + | <br><DT><span>1.4 Add 250uL of Solution II. Invert several times until you get a clear lysate.</span> |
<br><DD>1.4.1 Once Solution II is added, do not let it sit for more than 5 minutes! | <br><DD>1.4.1 Once Solution II is added, do not let it sit for more than 5 minutes! | ||
− | <br><DT> | + | <br><DT><span>1.5 Add 350uL of Solution III. Invert several times until a white precipitate forms. Centrifuge at 13,000xg or 17,900rcf for 10 minutes. A compact white pellet should form at the bottom of the tube.</span> |
− | <br><DT>1.6 Insert a mini column into a 2mL collection tube. | + | <br><DT><span>1.6 Insert a mini column into a 2mL collection tube.</span> |
− | <br><DT> | + | <br><DT><span>1.7 Transfer the clear supernatant into the mini column using a micropipette. Centrifuge at the maximum speed (13,000xg) for 60 seconds. Discard the filtrate and reuse the collection tube.</span> |
<br><DD>1.7.1 Be careful not to get any parts of the pellet! Tilt at an angle with the pellet at the top when micropipetting is advisable. | <br><DD>1.7.1 Be careful not to get any parts of the pellet! Tilt at an angle with the pellet at the top when micropipetting is advisable. | ||
<br><DD>1.7.2 Think about what you are discarding versus what you want to keep! | <br><DD>1.7.2 Think about what you are discarding versus what you want to keep! | ||
− | <br><DT> | + | <br><DT><span>1.8 Add 500uL of the HBC Wash Buffer diluted in isopropanol. Centrifuge at maximum speed (13,000xg) for 60 seconds. Discard the filtrate and reuse the collection tube.</span> |
<br><DD>1.8.1 All wash buffers will be centrifuged for 1 minute. | <br><DD>1.8.1 All wash buffers will be centrifuged for 1 minute. | ||
− | <br><DT> | + | <br><DT><span>1.9 Add 700uL of the DNA Wash Buffer diluted in ethanol. Centrifuge at maximum speed (13,000xg) for 60 seconds. Discard the filtrate and reuse the collection tube.</span> |
− | <br><DT> | + | <br><DT><span>1.10 Centrifuge the empty mini column at the maximum speed (13,000xg) for 2 minutes to remove the ethanol.</span> |
− | <br><DT> | + | <br><DT><span>1.11 Transfer the mini column to a nuclease-free 1.5mL microcentrifuge tube.</span> |
− | <br><DT> | + | <br><DT><span>1.12 Add 50uL of Elution Buffer (or sterile deionized water). Let it sit in room temperature for 60 seconds. Centrifuge at maximum speed (13,000xg) for 60 seconds.</span> |
− | <br><DT> | + | <br><DT><span>1.13 Store eluted DNA at -20℃.</span> |
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Revision as of 15:51, 19 October 2016
Experiments
Workflow
1. Miniprep/Nanodrop
2. Digest
3. Gel
4. Ligation
5. Transformation, Plate
6. Colony PCR (Screening)
7. Gel
8. Inoculate correct colony to a liquid culture.
Materials:
Miniprep: grown culture, microcentrifuge, 2 1.5mL microcentrifuge tubes, mini column and collection tube, Solution I, Solution II, Solution III, HBC Wash Buffer, DNA Wash Buffer, Elution Buffer, micropipette and tips
Nanodrop: nanodrop machine, miniprepped DNA, Kimtech wipes, micropipette and tips
Digest: miniprepped DNA, dH₂O, 10X RE-Mix, standard restriction enzyme, micropipettes and tips
Gel: agarose gel (make one if necessary), 1X TAE Buffer, power supply, chamber and electrodes, ladder, micropipette and tips, DNA
Ligation: vector, parts 1 and 2, ligase buffer, ligase, Antarctic phosphatase, microcentrifuge tube, ice, micropipette and tips
Transformation: ice, ligation mixture, competent cells, incubator, LB media, microcentrifuge tubes, micropipette and tips Plate: agar plate, micropipette and tips, beads
Colony PCR: dH₂O, buffer, VF₂, VR, Q5 polymerase, dNTP, DNA dilution, micropipette and tips, PCR tubes, thermocycler, ice
Gel: agarose gel (make one if necessary), 1X TAE Buffer, power supply, chamber and electrodes, ladder, micropipette and tips, DNA
Inoculate: LB media, dilution, micropipette and tips