Lab 5: PROCEDURE

Purification of the DNA

1. Add 1.4 mL of well-mixed culture to a microfuge tube. If your culture has settled, you can vortex the tube.
2. Spin for 1 minute at 8000 rpm in microfuge to pellet the bacteria. Discard the supernatant into 10% bleach (you can add 10% bleach to a beaker and discard supernatant into it).
3. Resuspend the bacterial pellet in 250μL of Buffer P1 – make sure the pellet is completely resuspended in the buffer either by vortexing or pipetting up and down.
4. Add 250 μL of Buffer P2. Mix by inverting the microfuge tube 4-6 times. Do not vortex or you will shear the genomic DNA. The solution should be viscous and slightly clear. Incubate for 3 minutes at room temperature. Do not exceed 5 minutes.
5. Add 350 μL Buffer N3. Mix by inverting the microfuge tube 4-6 times.
6. Spin the tube for 10 minutes at 13,000 rpm in the microfuge. Be nice to your podmates and wait until everyone is ready to start your spin!
7. Obtain a purification column in a collection tube. Apply the supernatant to the column and spin the tube containing the column for 1 minute at maximum speed. Discard the liquid that flows through the column into the collection tube (you can use a separate beaker or washer bucket to collect the flow through. Do not use the same beaker that has the bacterial supernatant, as bleach reacts with some chemicals in the flow through).
8. Attach the clear collection tube back to the column.
9. Add 750 μL PE Buffer and spin the column + tube for 1 min. at maximum speed. Discard the liquid that flows through the column. Attach the clear collection tube back to the column.
10. Spin the column + tube again for 1 min. at maximum speed. Discard the flow through liquid.
11. Transfer the column (without the collection tube) to a fresh microfuge tube.
12. Add 30 μL EB to the center of the column and let it sit for 1 minute at room temperature.
13. Spin the microfuge tube containing the column (with the added EB) for 1 minute at maximum speed. The liquid that flows through contains your DNA.
14. In order to get maximum recovery, add the flow-through back onto the column, incubate for 1 min. at room temperature and then spin for 1 min. at maximum speed.
15. Measure DNA concentration of all of your samples using the nanodrop spectrophotometer.

Restriction digest of the purified DNA

• Digest ~100 ng of each purified DNA sample. You will also digest negative (pETblue2 vector) and positive (pETblue2 with the HCAII insert) control DNA samples. Use a master mix for the digests. If your DNA concentrations are very high, you may need to dilute them. Think about how to do these dilutions such that your Master Mix is easier. You will share the control digests with a partner – so discuss with the person sitting next to you who will do them. You can even make a big Master Mix with your partner that would include the shared controls and all of your samples (See Lab 5: BACKGROUND for master mix example).

Restriction Digest:                                          Master Mix:

___ μL DNA

___ μL H20                                                ___ μL H20

1 μL 10x Buffer (CutSmart)                        ___ μL 10x Buffer (CutSmart)

0.5 μL enzyme                                             ___ μL enzyme

—————————————                            —————————————

10 μL final volume

Per Digest:

___ μL Master Mix

___ μL DNA

___ μL H20 (if necessary)

—————————————

10 μL final volume

• After setting up all of your digests, mix them well by gently flicking the tubes and then spin them in your nanofuge (the smallest centrifuge) for a couple of seconds to get all of the liquid to the bottom of the tube.
• Incubate your reactions at 37°C for 30 minutes.
• As soon as you start your digest, pour your gel! Pay attention to which comb you use when pouring the gel – how many samples will you have to load?

Agarose gel

Run all 10 μL of your digests on a 1% agarose gel to determine whether any of your minipreps contains the desired clone. You will also run a 10 μL sample containing uncut plasmid (one per gel). Be sure to include a ladder. Refer back to the lab 3 protocol for details on running gels: Lab 3: PROCEDURE

Future Steps

In a research lab, after running your gel you would identify which miniprep(s) appear to contain the clone you want. You would then have the purified DNA sequenced. It is common to sequence more than one potential clone to ensure that at least one has the correct sequence. Once a clone with the correct sequence has been confirmed, it would be transformed into an E. coli strain suitable for protein expression. Due to time and financial constraints in the 551 lab, the sequencing of clones and transformation into Tuner(DE3) E. coli cells will be done for you.

Clean up

• Dispose of bacterial culture tubes, columns, tubes to the biohazard bag.
• Dispose of the small/inner biohazard bag to the big biohazard bag. Replace with a new small biohazard bag (can be obtained at the TA table)
• Dispose of or put back reagents (see blackboard for details)
• Dispose of liquid in the drain (bacteria supernatant must sit in the bleach solution for at least 30 minutes before dispose down the drain)
• Put everything from the drawers back to the corresponding drawers. If supplies (such as beakers) are used to hold liquid, wash the supplies and leave them on the bench to dry. Do NOT dry them on the drying rack above the sink.
• Nothing needs to be saved in the freezer box at the end of this lab