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If at first you don't succeed, you are running about average.
M.H. Alderson

Day 2: DNA verification and DNA assembly


Assignments Due

Discussion

Overview of Experiment

BioBricks can be joined using standard cloning techniques and a process called BioBrick Standard Assembly. After analysis of the digests from day 1 on a 1% agarose gel, the digested vector and the insert DNA are gel purified.  The insert and vector are ligated, and competent bacterial cells are transformed with the ligation reaction. Transformations are plated on Luria Bertani (LB) agar plates containing 50 µg/ml ampicillin (Amp).

Background

Agarose gel analysis of restriction digests

Gel casting and running (each team pours two gels):

Caution

Agarose can become superheated and violently boil over. Exercise caution when heating. Swirl flask occasionally during heating. Heat until close inspection reveals that the agarose is 100% dissolved. Undissolved agarose will appear as little flecks that look like Lilliputian contact lenses.

Health Hazard

Ethidium bromide is a powerful mutagen and is moderately toxic and should be handled with care. WEAR GLOVES when handling contaminated equipment or solutions containing ethidium bromide. Confine the compound to the restricted area. Use plastic wrap to protect equipment and surfaces from being contaminated.

Note: Concentrated ethidium bromide solutions should be decontaminated. One method is to treat 0.5 µg/ml staining solutions of EtBr with 1 g/liter activated charcoal, filter and incinerate the residue. Slurries of activated charcoal can be used to decontaminate surfaces (see Maniatis et al, (1989) for additional methods of decontamination).




1.

Prepare gel tray as diagrammed using an adjustable gel caster.  Level the tray using a bubble level.

2.

Flasks of completely melted 1% agarose in 1X TBE have been prepared and the melted solution is incubating at 50°C. Carefully pour melted agarose (60 ml) into a beaker. Remember the solution is hot!!

3.

After the instructor adds EtBr to the gel, gently swirl the agarose. (Let the instructor know when you are ready to add the EtBr to your gel.)

4.

Immediately, pour the melted agarose into the level casting tray. Use a pipet tip to push bubbles towards the bottom of the gel.

5.

Let the tray cool until gel is translucent (takes at least 20 minutes). CLEAN UP ANY DRIPS ON THE BENCH AND RINSE THE BEAKER WITH RO-H2O!

6.

Prepare samples as described below. For agarose gels it is advisable to load the same volume into each well. Some samples may need to be diluted with water or TE to achieve this.

7.

Turn lever on the caster to break the seal. Carefully remove comb and place casting tray into the electrophoresis box for running. Fill unit with 1X TBE buffer to ~ 1 mm above gel. Pour buffer carefully onto the center of the gel to prevent the gel from sliding off the tray.

8.

Carefully load 10 µl of the DNA ladder (see below) into a well; load all of each plasmid DNA sample into different wells. Record the order of the samples in your notebook. Do not press the tip into the bottom of the well while loading--allow the sample to sink there.

9.

Position the lid and connect the electrodes in the correct orientation.

10.

Run gel at 130 V for ~ 30 min.
The 500 bp standard will run just behind the dark blue dye front, and smaller fragments that run ahead of the dye may not be visible in this type of analysis.

CAUTION: Lethal voltages are present while the power supply is "ON." Do not touch the gel or buffer until the electrodes are disconnected.

11.

WEAR GLOVES. Place casting tray with gel onto a paper towel and carefully carry to the photography area. DO NOT spread EtBr outside the designated area!! From this point forward, assume that your gloves are contaminated with EtBr. Do not touch anything that is not supposed to be contaminated.

Re-emphasize: Wear gloves and DO NOT spread EtBr outside the designated area!!

Place gel onto a sheet of plastic wrap on the transilluminator. CAUTION: The gel is still laden with EtBr and should be handled only with gloved hands. Scrupulously avoid all skin contact with the gel. Do not remove the gel from the designated EtBr bench. A waste container is provided there.

12.

Photograph the gel and compare the observed bands to the standards (see below).

13.

Locate the insert and vector DNA bands on the agarose gel using long wave (>300 nm) UV light.

Wear safety glasses or a face shield to protect your eyes and minimize exposure time to skin.

14.

Excise the DNA from the gel using a razor blade or scalpel (use a new one for each piece of DNA; dispose of in a sharps container).

cut as small a piece of agarose as possible -- trim off excise gel around the band

15.

Transfer each gel piece to a sterile 1.5 ml tube; proceed with gel purification of DNA


Sample preparation:


DNA standards:

Quick-Load® 1 kb DNA Ladder (New England Biolabs, Ipswich, MA): load 10 µl (0.5 µg) per lane



Expected products:


UVP BioDoc-It™ System System Components

Avoid doing anything that would intentionally contaminate the transilluminator or camera with EtBr. For instance, do not lay gels directly on the transilluminator, but always on plastic wrap. Do NOT contaminate the equipment (door knob, camera, printer, etc.)--REMOVE your gloves BEFORE working with the camera and gel documentation system.

  1. Position the gel in the center of the UV transluminator and smooth out the wrinkles in the plastic wrap.
  2. Close the darkroom cabinet's door and turn on the transilluminator.

    NOTE: the transilluminator will cut off if the darkroom door is opened.

  3. If the image on the LCD screen is dim, press the "+" button on the keypad controller; each time the "+" button is pressed, the CCD camera is set to the next increasing exposure time (there are 17 steps from 0.1 sec to 10 sec).

    NOTE: For more detail but a gray background, increase exposure time; for less detail and black background, decrease exposure time.

  4. Rotate the zoom adjustment (MIDDLE ring) on the lens so that the image is the appropriate size.
  5. Press the "Capture" button to hold the image on the screen.
  6. Press "Print" on the thermal printer in order to print the image; press AND hold copy/feed to advance the paper to cut the picture.

Dispose of the gels in the Biohazard Waste Box.
Do NOT put paper towels, plastic wrap, or gloves in this waste box--ONLY the gels.


Gel purification of digested DNA

For the E0840 digest, we want only the 878 bp insert band; for the digested and phosphatase-treated R0040, we want only the cut vector (i.e., we do not want any uncut plasmid carrying over into the ligation reaction). After cutting out the pieces of agarose containing the appropriate DNA bands, the agarose is melted and the DNA is recovered using a mini spin column.

PROTOCOL (adapted from Zymoclean Gel DNA Recovery Kit™):
  1. Pulse spin for ~ 10 sec to "pellet" the agarose piece containing the insert band from E0840 and the piece containing the vector band from R0040 (for estimation of gel volume)
  2. Add 3 volumes of ADB Buffer™ to each volume of excised agarose
  3. Incubate at 50-55°C until the agarose is completely dissolved (5-10 minutes)
  4. Add the melted agarose solution to a spin column in a collection tube
  5. Centrifuge at 16,000 x g for 30 seconds and discard column flow-through
  6. Add 200 µl wash buffer (contains ethanol) and centrifuge as in step 5 (do not discard flow-through)
  7. Repeat the wash and centrifuge as in step 5
  8. Discard flow-through and centrifuge at 16,000 x g for an additional 30 seconds to remove the last drop of wash from the column (residual ethanol will inhibit DNA elution)
  9. Put column in a sterile 1.5 ml tube
  10. Add 10 mM Tris-HCl, pH 8.5, 0.1 mM EDTA (same components as Zyppy™ Elution Buffer) to center of column
  11. Centrifuge at 16, 000 x g for 30 seconds to elute DNA
    ***The gel-purified DNA can be used directly in ligations, digests, PCR, etc.***
  12. Proceed to DNA ligation

DNA ligation

Vector: SpeI / PstI digested BBa_R0040, ~2100 bp
Insert: 878 bp fragment from XbaI / PstI digest of BBa_E0840

A. Estimation of concentrations of insert and vector

If a DNA sample is too dilute to measure at 260 nm or is contaminated with other compounds that absorb in the UV range, the amount of DNA present can be estimated from the intensity of ethidium bromide fluorescence. Since the amount of DNA in a solution is proportional to the fluorescence emitted by ethidium bromide, the DNA quantity in an "unknown" solution can be estimated by comparing its level of fluorescence with the intensity of known amounts of DNA.

You need to estimate concentration of the gel-purified insert and vector fragments.  Compare the intensity of the "unknown" DNA (e.g., 2X as bright) to a band in the 1 kb ladder to estimate the DNA concentration of your sample (ng of DNA for each size standard are given in the Table on the manufacturer's insert). Consider the initial volume that contained the DNA; the volume loaded onto the gel is not critical to your calculation of concentration
  1. Preparation of samples:
    a. Put 2 µl digested/phosphatased/gel-purified vector or 2 µl gel-purified insert in a sterile tube
    b. Add 8 µl NF-water
    c. Add 2 µl 6X LB
  2. Load all of each sample into the mini gel and load 1 kb DNA ladder (10 µl)
  3. Run a 1% agarose gel at 130 V for 20-30 min
  4. Photograph the gel
  5. Compare the relative intensity of staining of the unknown with a comparably sized band in the 1 kb ladder and estimate the DNA concentration of your original samples
B. Protocol from Quick Ligation™ Kit (New England Biolabs):
  1. Thaw 2X Quick Ligation Reaction Buffer (2X QLRB) [132 mM Tris-HCl, pH 7.6 @ 25°C; 20 mM MgCl2; 2 mM DTT; 2 mM ATP; 15% polyethylene glycol (PEG 6000)] ON ICE and mix thoroughly; the ligase buffer contains ATP and Mg2+ necessary for the reaction
  2. Set up TWO ligation reactions in 1.5 ml tubes (per team):
  3. Add 10 µl of 2X QLRB to each reaction and mix thoroughly
  4. Add 1 µl of Quick T4 DNA Ligase (lig) to each reaction and mix thoroughly
  5. Centrifuge brielfy and incubate the reactions at room temperature for 5 min
  6. Chill on ice and proceed to transformation

Bacterial transformation of E. coli

To amplify plasmid DNA for manipulation and analysis, we are going to transform bacteria with the ligation reaction. The cell strains we use are either sensitive (S) or resistant (R) to the antibiotic tetracycline (Tet); these cells were harvested at early - mid log phase and were made competent for transformation (Z-Competent™ cells were prepared by the instructor according to the manufacturer’s protocol).

Keep in mind that transformation efficiency when using ligated DNA is greatly reduced compared with using plasmid DNA.
Several control transformations are performed today:
1) plasmid DNA = ensures competent cells are good
2) digested/phosphatased/gel-purified vector ONLY = gives "background" for UNCUT vector
3) digested/phosphatased/gel-purified vector PLUS ligase = gives "background" for vector recircularization


PROTOCOL [adapted from the Z-Competent™ E. coli Transformation Kit & Buffer Set (Zymo Research Corp., Orange, CA)]:
  1. Obtain 0.1 ml aliquots of “Z-Competent™” TetS strain (GNB8385K) or TetR strain (GNB824) cells and place on ice

    You must be extremely gentle when working with competent cells. These cells are highly sensitive to temperature changes and/or mechanical lysis. Mix cells by gently tapping the tube or swirling with a pipet tip, not by pipetting up & down or vortexing.

  2. After cells thaw, as a team, prepare the following transformations:
    Three teams should perform the positive control transformation:
  3. Mix gently and incubate samples on ice for 5 minutes
  4. Pipet 100 µl of the (+) plasmid DNA transformation onto an LB-Amp (50 µg/ml) plate (prewarmed to 37°C)
  5. Pour 10 - 20 sterile solid glass beads onto the plate, set the plate on the benchtop, and "shake" plate back and forth on the bench top for a few seconds; invert plate to pour off beads (collect in a large beaker -- these can be cleaned, autoclaved, and reused)
  6. Pipet 100 µl of the (-) R0040 ONLY transformation on a 2nd LB-Amp plate (prewarmed to 37°C)
  7. REPEAT step 5
  8. Pipet 100 µl of the (-) R0040 plus ligase transformation onto a 3rd LB-Amp plate (prewarmed to 37°C)
  9. REPEAT step 5
  10. Pipet 100 µl of the R0040+E0840 transformation onto a 4th LB-Amp plate (prewarmed to 37°C)
  11. REPEAT step 5
  12. Let the plates sit 5 minutes at room temperature so that the liquid absorbs into the agar
  13. Incubate the plates (upside down) overnight at 37°C

Copyright, Acknowledgements, and Intended Use
Created by B. Beason (bbeason@rice.edu), Rice University, 21 November 2007
Updated 3 July 2014