Difference between revisions of "Team:Lambert GA/Experiments"

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

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. (Original protocol called for 10,000xg for 1 minute, but the speed and time above seemed to produce better results.)
1.2.1 Original protocol called for 10,000xg for 1 minute, but the speed and time above seemed to produce better results.\
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.
1.4 Add 250uL of Solution II. Invert several times until you get a clear lysate.
1.4.1 Once Solution II is added, do not let it sit for more than 5 minutes!
1.5 Add 350uL of Solution III. Invert several times until 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.
1.6 Insert a mini-column into a 2mL collection tube.
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.
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.
1.7.2 Think about what you are discarding versus what you want to keep!
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.
1.8.1 All wash buffers will be centrifuged for 1 minute.
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.
1.10 Centrifuge the empty mini column at the maximum speed (13,000xg) for 2 minutes to remove the ethanol.
1.11 Transfer the mini-column to a nuclease-free 1.5mL microcentrifuge tube.
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.
1.13 Store eluted DNA at -20℃.

Nanodrop
2.1 Vortex before nanodrop.
2.2 Wipe down the nanodrop machine with Kimtech wipes to make it sterile.
2.3 Set the program to analyze nucleic acids [because you are dealing with plasmid DNA].
2.4 Do a blank test to ensure that the platform is sterile.
2.5 Load 1uL of the miniprepped DNA onto the platform.
2.5.1 (Have steady hands. The sample needs to be in the center for best results.)
2.6 Click “measure” on the nanodrop for analysis.
2.7 Write down measurements for the concentration of DNA (in ng/uL), A260, A280, 260/280 (should be around 1.8), and 260/230 (should be around 2.1).

Digest
3.1 Dilute up to 1ug DNA to 17uL with dH₂O.

3.1.1 Take concentration of DNA from nanodrop and convert from ng/uL to ug/uL. Next, set up a proportion to find out how many uL you need to get 1 ug of DNA.
3.1.2 20uL (total reaction) - 2uL RE-Mix - 1uL standard enzyme = uL dH₂O

3.2 Use a microcentrifuge tube to put the reaction in. Put in the contents in this order: water, DNA, enzymes.
3.2.1 Add 2uL of the 10X RE-Mix and 1uL of the standard enzyme.
3.2.1.1 E and X = 10X RE-Mix 3.2.1.2 S and P = standard enzymes
3.3 Incubate at 37℃ for 1 hour for standard enzymes, then at 80℃ for deactivation.