Lab 5: PROCEDURE

Starting overnight cultures

Note that this needs to be done on the afternoon before the main experiment, so that the E. coli cultures can grow overnight.

Take a look at the plates from last lab and identify the one with the most colonies that are potentially positive clones. This is the plate you will use.

This video demonstrates the procedure for picking colonies and starting overnight cultures:

• Label three test tubes with your name
• Add 2 mL LB with 50μg/mL ampicillin to each test tube
• With a sterile toothpick, touch a single bacterial colony and drop the toothpick into 2mLs
  • Repeat for a total of three times; i.e. each test tube will contain a single colony
• Incubate at 37°C shaking overnight.

Purification of the DNA

Note that this protocol is similar to the DNA purification done previously, but slightly different. In the previous lab, you started with PCR product, whereas here we are starting with whole cells (which need to be pelleted and lysed, and leave debris that must be removed).

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 bleach.
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 min. at 13,000 rpm. (~ 17,900 x g) 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 min. at maximum speed. Discard the liquid that flows through the column into the tube. You should re-use the clear collection tube.
8. Add 750 μL PE Buffer and spin the column + tube for 1 min. at maximum speed. Discard the liquid that flows through the column. You should re-use the clear collection tube.
9. Spin the column + tube again for 1 min. at maximum speed. Discard the flow through liquid.
10. Transfer the column to a fresh microfuge tube.
11. Add 30 μL EB to the center of the column and let it sit for 1 minute at room temperature.
12. 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.
13. 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.
14. Measure DNA concentration of all of your samples using the nanodrop spectrophotometer.

Restriction digest of the purified DNA

We have also seen a restriction digest in lab before. This case is slightly more complicated because rather than simply adding enzyme to DNA, as we did with DpnI, this enzyme requires a specific buffer, and we want all the DNA samples to be at about the same concentration.

• 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.

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? Remember that you are sharing your gel with a partner – so you have to count each of your three samples, plus the two digested controls, and one uncut control, plus a ladder. Note that all of the controls are shared between partners.

Agarose gel

Run all 10 μL of your digests on a 1% agarose gel to determine whether any of your minipreps contain 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 2 protocol for details on running gels: Lab 2: PROCEDURE Part2

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.