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<h3 style="text-decoration: none; color: #D49AE6;"> Protocol: </h3> <br> | <h3 style="text-decoration: none; color: #D49AE6;"> Protocol: </h3> <br> | ||
− | + | <b> 1. Miniprep (using Omega protocol)</b><br> | |
1.1 Grow 1-5mL culture overnight in a 10mL-20mL culture tube. | 1.1 Grow 1-5mL culture overnight in a 10mL-20mL culture tube. | ||
<br> 1.2 Centrifuge at 2500xg for 5 minutes at room temperature. Decant or aspirate and discard the culture media. | <br> 1.2 Centrifuge at 2500xg for 5 minutes at room temperature. Decant or aspirate and discard the culture media. | ||
− | <br> 1.2.1 Original protocol called for 10,000xg for 1 minute, but the speed and time above seemed to produce better results. | + | <br> <style="text-decoration: none; color: #D49AE6;">1.2.1 Original protocol called for 10,000xg for 1 minute, but the speed and time above seemed to produce better results. |
<br> 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. | <br> 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. | ||
<br> 1.4 Add 250uL of Solution II. Invert several times until you get a clear lysate. | <br> 1.4 Add 250uL of Solution II. Invert several times until you get a clear lysate. |
Revision as of 02:01, 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
Protocol:
1. Miniprep (using Omega protocol)
1.1 Grow 1-5mL culture overnight in a 10mL-20mL culture tube.
1.2 Centrifuge at 2500xg for 5 minutes at room temperature. Decant or aspirate and discard the culture media.