• Day 1
• Day 2
• Day 3
• Day 4
• Day 5
• Day 6

Resources

• *LAB SAFETY**
• Laboratory notebook
• Graphing tutorial
• Molecular biol tips
• Writing assignment
• Bios 211 web site
• Searching literature

Other links

• Bios 111 honor code
• Graphing quiz (rice only)

Bios 111 Day 4

Confirmation of Bacterial Transformant

Introduction

Referring to your previous notes, you will conduct plasmid DNA mini preps of transformants, restriction enzyme digests of plasmid DNA, pour, load, and run agarose gels, and calculate efficiency of transformation (EOT).

Background

Review the relevant background information for days 1-3.

Experimental Overview

• Plasmid DNA mini prep (individual)
• RE digests of plasmid DNA (individual)
• Agarose gel electrophoresis (pair)
• Counting colonies to determine EOT (pair)

A) Plasmid DNA mini prep

We're using the Zyppy™ Plasmid Miniprep Kit (Catalog No. D4036, Zymo Research Corp.) to isolate plasmid DNA from a 3 ml overnight (O/N) bacterial culture.  Each overnight culture arose from a single colony: a well-isolated colony on the LB-kan plate was lightly touched with a sterile toothpick or a sterile pipet tip; after twirling the tip in a 15 ml culture tube containing liquid LB plus kan, the tubes were incubated with shaking at 37°C overnight.  NOTE: in a “real” lab situation, you would save some of the overnight culture for streaking a plate and making a glycerol stock.

* Disposal of Waste: Discard bacterial supernatant in a small beaker; add bleach to 10% for 10 minutes before dumping into the sewer
* Collect contaminated tips and culture tubes in a beaker at your desk then rinse the beaker with bleach after dumping the waste in the clear Biohazards bag; these bags will be autoclaved prior to placement in the household trash
* After the bacteria are lysed, tips, vials, and other materials should be discarded in the regular trash

PROTOCOL (refer to your lab notebook)

B) Restriction enzyme (RE) digests of plasmid DNA

We are going to use RE digests to confirm the identity of the bacterial transformants.  You will set up a single digest with HindIII  and a double digest with HindIII/PstI.  Since we are not using the digested DNA for a ligation or some other reaction where residual restriction enzyme could interfere, we do not need to clean-up the DNA; ALL of the digest reaction will be loaded onto an agarose gel for analysis. 

PROTOCOL (refer to your lab notebook)

Our enzymes are from New England Biolabs or Promega; be sure to record the units/µl for each enzyme you use. Buffer components (1X) for each buffer can be found in the catalogs.

• Put 10 µl plasmid DNA in three sterile 1.5 ml tubes and label tubes
• UC (uncut control reaction)
• HindIII (single digest)
• HindIII/PstI (double digest)
• Add 2.5 µl NEBuffer 2 to each tube
• Add 2.5 µl 10X BSA to each tube
• Add NF water for a TOTAL reaction volume of 25 µl
• Remember to account for the volumes of plasmid DNA, buffer, BSA, and enzyme(s)
• Add 1 µl of the appropriate enzyme(s)
• Mix and incubate as on lab day 1

C) Agarose gel electrophoresis (each pair pours gel while RE digests are incubating at 37°C)

PROTOCOL

***HEALTH HAZARD***

•Ethidium bromide is a probable carcinogen and should be handled with care.  Wear gloves when handling contaminated equipment or solutions containing ethidium bromide.  Confine the compound to the restricted area.  After soaking the gels, do NOT remove gels or other contaminated materials from the photography area.  Use plastic wrap to protect equipment and surfaces from being contaminated.

•Ethidium bromide solutions should be decontaminated.  One method is to treat 0.5 mg/ml staining solutions of EtBr with 1 g/liter with 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).

Remember: Wear gloves and DO NOT spread EtBr outside the designated area!!

1. Rinse the gel tray and comb(s) then prepare gel tray as diagrammed above. Tape the ends of the casting tray as indicated.  Level the tray using a bubble level.
2. Flasks of completely melted agarose in 1X TBE have been prepared and the melted solution is incubating at 50-55°C. Hold the hot flask with a folded paper towel and carefully pour ~60 ml melted agarose into a beaker. Remember the solution is hot!!
3. After the instructor adds EtBr to the agarose, gently swirl the agarose. (Let the instructor know when you are ready for EtBr.)
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. Allow the tray to cool until gel is translucent. This will take at least 20 minutes. CLEAN UP ANY DRIPS ON THE BENCH AND RINSE THE FLASK!
6. Prepare samples
• Digest reactions: add 5 µl 6X LB to each sample
7. Carefully remove comb and tape and place casting tray into the electrophoresis box for running. Fill unit with 1X TBE buffer to ~ 1 mm above gel. Pour carefully onto the center of the gel to prevent the gel from sliding off the tray.

• Load ALL of each sample as on lab day 2
• Load 10 µl Quick-Load 1 kb DNA Ladder
• Run at 130 V for ~ 45 minutes
• Obtain a picture of the gel as on lab day 2

ATTENTION: Avoid doing anything that would unintentionally 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.

D) Efficiency of transformation (EOT) = # colonies / per µg DNA

EOT is calculated by counting the number of colonies that grow on selective media following transformation and dividing by the total µg DNA used in the transformation. Dilutions must be calculated to determine the amount of DNA present in the volume of transformed culture placed on each plate.

Homework Assignments

Prepare all of the homework assignments in your laboratory notebook and turn in the duplicates at the beginning of the next laboratory session.

1. Restriction Digest Analysis

Use the plasmid map and the picture of your gel from today to obtain the following information:

1. From the plasmid map, predict the theoretical number and sizes of the restriction fragments for both the single and double digests. Give the rationale for your answer.
2. Plot log10 (# base pairs) versus distance migrated from the well and draw a standard curve in your lab notebook to estimate the sizes of the restriction fragments
3. Compare the experimental results with the expected sizes: do they agree? If not, suggest possible reasons for the discrepancy.

2. EOT Calculation

Using the number of colonies from your plates, calculate the EOT for your experiment.  Show all
work in your lab notebook.

• Assume ~ 5 ng gel purified PstI-digested DNA was used in the ligation reaction
• Remember to account for dilution of the plasmid DNA during the transformation and plating of cells

Reminder

The final version of the materials and methods section is due the last week of labs on your lab day. It is to be a complete write-up of materials and methods for days 1-4. Remember to double space with 1 inch margins and use either Times or Times New Roman font (12 point). Please attach your graded draft to the back of your revised methods section.