• 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 1

Isolation and Manipulation of Plasmid DNA

Introduction

Today's laboratory procedures look like an awful lot of work, but in fact the protocols go very fast. The challenge is to conduct each procedure exactly as instructed. Molecular biology requires faithful attention to details. Deviation from an established protocol almost always result in failed preparations.

• Plasmid DNA mini prep
• Restriction enzyme (RE) digests of plasmid DNA
• Preparation of Luria-Bertani (LB) plates

All of these procedures are "staples" in a laboratory that works with DNA. Your experience with these methods will be greatly appreciated if you take on a project in such an environment.

Background

PLASMID DNA

Plasmids are small circles (usually less than 15 kb) of double stranded DNA maintained in some bacteria because they confer an advantage to the cells such as resistance to an antibiotic. The ease with which this DNA can be isolated and manipulated accounts for the widespread use of plasmids in molecular biology for a variety of tasks (e.g., protein expression). Although plasmids occur naturally, the ones used in research have been engineered to prevent natural transfer between bacteria to reduce the spread of antibiotic resistance.

Plasmids used for protein expression in E.coli need to have:

1. Replication origin
2. Selectable marker
3. Promoter
4. Ribosomal binding site (RBS)
5. Multiple cloning site (MCS)

RESTRICTION ENZYMES

Restriction enzymes cleave the phosphodiester bonds in each strand of double-stranded DNA. The cleavage may be at adjacent sites leaving a “blunt end,” or the cut may be offset by 1 to 4 bases, leaving either a 3' overhang or a 5' overhang of a single strand. The offset cleavage yields "sticky end" cuts.

Restriction enzymes are obtained from many prokaryotes and about 1500 enzymes with known sequence recognition sites have been isolated. Naming these endonucleases follows a system proposed by Nathans and Smith. Each name contains at least one capital letter and two small letters followed by a Roman numeral. The letters are initials of the genus and species of origin and the number represents the number of enzymes discovered in the organism. (Historically the numeral identified the protein peak in which the enzyme eluted during chromatography.) Additional information may be added as a letter. For EcoRI, the R indicates the particular strain of E. coli.

A few buffer conditions suit nearly all the restriction enzymes but no single buffer allows activity of every enzyme. Suppliers of enzymes always provide a reaction buffer (10x concentrate) that is optimum for the enzyme. Components of the 1x buffer usually are 10-100 mM Tris at pH 7.3 to 8.5, various levels of salts like KCl and NaCl (10 to 150 mM), 10 mM Mg(2+), 2 mM beta-mercaptoethanol. Sometimes 0.01% Triton- X100 (a detergent) and bovine serum albumin are included as a stabilizers. (Alternatively, swine skin gelatin can be used and offers the advantages that it is stable to autoclaving and costs about 1/15 as much as BSA.)

Since restriction enzymes can require different buffer conditions, some strategy must be used to do double digests. The preferred method is to simultaneously digest with both enzymes in a compatible buffer. This method can be used even if one enzyme is not fully active (e.g., 75% active). More of one enzyme can be added (e.g., 1 U of enzyme A + 1.33 U enzyme B) for equal cutting efficiency. There are limits to the excess enzyme due to increased glycerol in the reaction that can reduce specificity of some enzymes.  An alternative method is to digest with the "low salt" enzyme then add more buffer and the "high salt" enzyme to complete the digest. This obviously doubles the time required for digestion. In extreme cases the DNA can be precipitated after one digest and dissolved in the second digest buffer. Digests are carried out at 37 degrees C unless otherwise noted for the enzyme.

Miscellaneous information on restriction enzymes

• By definition, a unit of restriction enzyme will completely cleave 1µg of Lambda DNA (or other substrate DNA) in one hour in the recommended buffer and temperature.
• Reaction volumes should be 25-50 µl and the amount of enzyme added should not exceed 10% of the volume due to the glycerol content. The amount of DNA in a digest should not exceed 250 µg/ml because the increase in ionic compounds in the DNA preparations will decrease the efficiency of cleavage.
• Restriction enzymes are most stable when stored in a non-cycling -20 degrees C freezer. (Frost-free freezers are not recommended.) Most enzymes are stored in 50% glycerol and therefore are not frozen solid, allowing removal of the enzyme without thawing. Always keep the enzyme solution in a freezer block (e.g., a “Stratacooler”) and return it to the freezer as quickly as possible.
• Catalogues from suppliers represent an accessible (and free) source of valuable information on restriction enzymes. A wealth of information is usually found in table form in appendices in the back of the catalogue or near the pages listing the restriction enzymes. These tables give buffer concentrations, recognition sequences, E. coli strains and genotype tables, and more. New England Biolabs and Promega are exceptionally efficient at providing information; additionally, both companies provide several useful web-based resources.

Experimental overview

Today’s procedures involve isolating and manipulating plasmid DNA.  On some procedures you will work as an individual; on others you will work with a partner.  Perform the procedures in the order given below.  Make sure that you use the appropriate pipettor and set the volume correctly—if you’re unsure, then ask.  Record all procedures and data in your lab notebook, indicating “who” performed a procedure step when you work as pairs; turn in copies of notebook pages at the end of the laboratory session.

• Plasmid DNA mini prep (individual)
• Restriction enzyme (RE) digests of plasmid DNA (individual)
• Preparation of Luria-Bertani (LB) plates (pair)

SPECIAL NOTE:  Record enough procedure details in your notebook during lab today so that you can repeat these procedures using your notebook as the ONLY resource.  Write the methods in your own words (i.e., do not just “copy” the steps from the web pages or handouts).

ADDITIONAL DETAILS:  for each centrifugation, record time, rcf (# x g), and temperature in your lab NB; ALL centrifugations performed in the microcentrifuges are at “room temperature.”

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.

• Disposal of Waste: Discard bacterial supernatant and collect contaminated tips in a small beaker; we will add bleach to 10% for 10 minutes before dumping into the trash and sewer
• Place culture tubes 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 (from the Zyppy™ Plasmid Miniprep Kit Instruction Manual)

1. Pipette 1.5 ml of bacterial culture (record the ID on the culture tube in your NB) into a sterile 1.5 ml microcentrifuge tube and centrifuge for 30 seconds at 16,000 x g (in a microcentrifuge); carefully pour the supernatant (liquid) into small waste beaker (a small amount of liquid can remain in the tube)
2. REPEAT step 1 (use the SAME microcentrifuge tube); after pouring off waste, remove remaining liquid using your pipettor
3. Add 600 µl nuclease-free (NF) water to the cell pellet and resuspend completely by gently pipetting up and down
   • make sure there are no “clumps” of bacterial pellet visible
4. Add 100 µl of 7X Lysis Buffer (Blue) and mix by inverting tube 6 times
• You must proceed to step 5 within 2 minutes (excessive lysis can denature plasmid DNA!)
5. Add 350 µl COLD Neutralization Buffer (Yellow), containing 100 µg/ml RNaseA, and mix thoroughly by inverting tube
• When neutralization is complete, sample turns yellow and a yellowish precipitate forms
• Invert sample 2-3 more times (to ensure complete neutralization)
6. Centrifuge at 16,000 x g for 4 minutes
7. Transfer supernatant to a Zymo-Spin™ II column (avoid disturbing pellet!)
8. Put column in a 2 ml collection tube and centrifuge for 15 seconds (maximum speed, press and hold the "short" button)
9. Discard flow-through (liquid waste) in sink and put column back into the collection tube
10. Add 200 µl Endo-Wash Buffer to column and centrifuge for 15 seconds at 16,000 x g (press and hold the "short" button)
11. Add 400 µl Zyppy™ Wash Buffer (containing ethanol) to the column and centrifuge for 30 seconds at 16,000 x g)
12. Transfer column into a sterile 1.5 ml microcentrifuge tube
13. Add 30 µl NF water directly to the column matrix (white circle) and wait one minute (room temperature)
14. Centrifuge 15 seconds at 16,000 x g (press and hold the "short" button) to elute plasmid DNA
15. Proceed to B)

B) Restriction enzyme (RE) digests of plasmid DNA

Our enzymes are from New England Biolabs or Promega; be sure to record the units/µl for each enzyme you use (information given on the manufacturer’s insert). Buffer components (1X) are listed on the manufacturer’s insert.

PROTOCOL

1. Put 10 µl plasmid DNA in a sterile 1.5 ml tube; label the tube “uncut” (CONTROL sample)
2. Put 10 µl plasmid DNA in a sterile 1.5 ml tube; label this tube “PstI” (RE DIGEST sample)
3. Add 5 µl nuclease-free (NF) water to the uncut control and 4 µl NF water to the digest
4. Add 2.5 µl NEBuffer 3 to each tube
5. Add 2.5 µl 10X bovine serum albumin (BSA) to each tube
6. Add 1 µl PstI to the digest
7. Gently flick tubes to mix and “pulse spin” (press and hold the “short” button for ~ 10 sec) samples
8. Incubate both samples at 37°C for at least 15 minutes in the “dry” heat block
9. Pulse spin samples
10. Store uncut DNA and PstI digest at -20°C in a “Stratacooler” (record the # in your lab NB)

C) Preparation of Luria-Bertani (LB) plates

***DEMONSTRATION BY INSTRUCTOR***

Although part C) is a demonstration by the instructor, you are still responsible for recording the procedure – media, antibiotic, labels on plates, etc. and a brief description of "how" you would pour LB-agar plates. If you do independent study there is a good chance that you will be preparing and using agar plates. The write-up that follows describes a procedure that you would follow if you were doing this part yourself.

LB plates, with the antibiotic kanamycin (Kan), will be prepared today for use on weeks 2 and 3.

Recipe for LB Agar (per liter):
1% Bacto-Tryptone = 10 g
0.5% Yeast extract = 5 g
NaCl = 5 g
Bacto Agar = 15 g

*adjust pH to 7.0 with NaOH and bring to 1 L; sterilize by autoclaving for 20 minutes at 121°C at 15 psi
**autoclaved media is always cooled to 50-60°C prior to the addition of antibiotics and some salts that are inactivated or precipitated by autoclaving; holding the agar at this temperature prevents congealing.

Sterile technique
Media can be contaminated by contact with non-sterile surfaces or by air borne organisms. Remove lids and coverings carefully avoiding contact with any part of the cover that may contact the media. Lids and coverings should be held with media side down at all times. Air borne contaminants are usually falling downward. Replace the coverings carefully so that the rim of the container makes contact only with sterile surface of the inside of the cap.  The use of a flame helps maintain aseptic materials. Working near a flame can decrease air borne contamination. The flame is also used to singe surfaces to maintain sterility. The mouth of the tube or flasks is passed through the flame before and after pouring. The cap or cover is also passed through the flame prior to replacing on the container.

Caution: The flame is used to singe the surfaces only. Do not hold the items in the flame to make them hot. Glass flasks, even Pyrex, can break from the heat or when the cooler media hits the hot surface.

Label the bottoms of 5 Petri dishes: indicate the types of plates (LB+Kan); also put your name or initials (everyone’s plates will be stored together). The bottoms are labeled because the lids can get separated. Also the plates are usually handled inverted.

1. Remove gloves
2. Turn on gas ~ halfway and light Bunsen burner with striker
3. Swirl large flask of LB-agar to mix contents well
4. Transfer ~ 125 ml of LB-agar to a sterile flask

***Use sterile technique - don't contaminate the media***

5. Add kanamycin (Kan) [STOCK = 50 mg/ml in water (stored at - 20°C)] to the LB agar in a 1:1000 ratio (1 µl of antibiotic for every 1 ml of solution)

***Show this calculation in your notebook***

6. Mix by swirling but avoid creating bubbles; keep the solutions sterile
7. Using sterile technique, pour 20-25 ml of LB-Kan agar into FIVE Petri dishes; add agar solution until the bottom of the dish is covered
• Work rapidly to pour the plates. The agar will begin to congeal in 5-10 minutes with the flask at room  temperature.
• The plates can be placed individually on the surface of the desk or left in a stack while pouring. The stack method saves bench space and is accomplished by lifting the stack of 4 empty plates by the lid of the bottom most plate. Agar is poured into the Petri dish and the stack is replaced. Move your hand to the second lid and lift the stack again and pour the second dish of agar. Repeat until all the plates in the stack are filled.
• If bubbles are present on the surface of the media, they should be eliminated by passing the flame of the burner quickly over the bubbles.
8. Turn off the gas!
9. Set the plates at room temperature until they are congealed; this usually takes 30-45 minutes but check that the agar is set in the center plates by gently shaking

*The instructor will put the plates in plastic and store the plates inverted at 4°C*

10. Pour any remaining media in the trash can and rinse out flask with water

Homework Assignments

Unless otherwise advised, please prepare all of the homework assignments in your laboratory notebook and turn in the duplicates at the beginning of the next laboratory session.

Make predictions: Using the plasmid map, predict the number of bands and the fragment size of the PstI digest.  Give the rationale for your answer.

Graphing tutorial and quiz

This laboratory course is about the "fundamentals." Among the most basic analytical skills you will need is the ability to produce an effective figure in the form of a graph. In fact, next week you will be introduced to the concept of a DNA standard curve. Before your next lab day (Day 2), please complete the graphing tutorial (also listed under "Resources," near the top of this page). When you are confident in your graphing skills, please submit the on-line quiz. The quiz, which will be included among your homework assignments, is to be sent before your second lab day.