Student examining a specimen in a microscope

Technical Resources

Biological, Environmental, and Earth Sciences

Laboratory Safety Guidelines

General Laboratory Practices

Maintaining proper laboratory records. Your laboratory records, and your ability to record and present key technical data in a meaningful way are crucial skills for the Biology Major.

Detailed Lab Notebook Instructions

Rules for keeping a laboratory book

1. Always write with a black (or blue) ink pen

2. Write everything you did and how you did it (in detail)

3. Write in real time (YOUR LAB NOTES ARE NOT MEANT TO BE PERFECT)

4. At the top of each page state the procedure used and cross-reference the procedure with the page number and notebook number if the procedure has already been written down.

5. If a mistake is made, cross it out with a straight line

6. Format:

a. Title, Date, and Page number

b. Summary of what was done (procedure used along with any changes in the procedure)

c. Results (written in RED ink). Figues should be accompanied by a descriptive legend

Lab meetings. Mandatory laboratory meetings are held every Friday afternoon following the departmental seminar. The start time for the meetings are typically 2:15 to 2:30; however, the time may be adjusted depending on circumstances. Every effort should be made to attend all lab meetings. At each meeting one lab member will be in charge of presenting his/her work (experiments, results, problems, etc.) to the rest of the group. Each presentation is followed by a question/answer period. Presentations begin each semester with those who have the longest tenure in the lab. 

 

Protocols 

Protocols by Application

 -A-

Amplification

Apoptosis

-B-

-C-

Cell Culture (Bacterial)

Cell Culture (Eukaryotic)

Cell Viability (Eukaryotic)

-D-

DNA Manipulation

-E-

-F-

-G-

Gel Electrophoresis

-H-

-I-

In Vitro Transcription

-J-

-K-

-L-

-M-

Modifying Enzymes

-N-

-O-

-P-

Protein Analysis

-Q-

-R-

Retrovirus Production and Transduction

Ribozymes

RNA Interference

RNA Manipulation

-S-

Stains and Staining

-T-

Transfection (Eukaryotic)

Transformation (Bacterial)

-U-

-V-

-W-

-X-

-Y-

-Z-

 

Reagents

 

-A-

 

Acrylamide gel for sequencing

29.42 grams Urea

10.5mL Acryl bis (19:1)

10mL TBE (1X)

25mL H2O

Mix all ingredients and filter through a Millipore filter unit. Add 330m L of 10% ammonium persulfate (APS) [e.g., 60m g APS in 600m L H2O]. Add 33m L TMED. Swirl to mix and pour immediately.

 

Agarose gel

Add the desired amount of agarose (Molecular Biology Grade) to a volume of running buffer (1X TAE or 1X TBE) sufficient for constructing a gel. For example, for 1% agarose add 1 gram of agarose per 100mL of buffer.

Melt the agarose in a microwave (swirl occasionally to ensure even mixing). Gel solution will be EXTREMELY HOT!

Replace the lost volume (due to boiling) with deionized water (NOTE: mixture must be preweighed).

Add 3m L 10mg/mL ethidium bromide to each 100ml of gel solution.

 

Alkaline Lysis Buffers, plasmid preps

Solution I

50mM glucose

25mM Tris-HCl, pH 8.0

10mM EDTA, pH 8.0

This solution can be prepared in batches of approximately 100ml, autoclaved for 15 minutes, and stored at 4° C.

Solution II

0.2N NaOH (freshly diluted from 10N stock)

1% SDS

Prepare fresh prior to use.

Solution III

60ml 5M Potassium acetate

11.5ml Glacial acetic acid

28.5ml dH2O

This solution is 3M with respect to potassium and 5M with respect to acetate.

Ref: Molecular Biology: A Laboratory Manual (1989) Eds: Sambrook et.al.

 

Alsever's Solution

Dextrose (20.5 grams)

Sodium citrate (8 grams)

Citric acid (0.55 grams)

NaCl (4.2 grams)

Dissolve ingredients successively in deionized water and autoclave at 15psi for 15 minutes. The pH of the sterilized solution should be 6.1. Add one part Alsever's solution to one part whole blood. Allow suspension to stabilize at 4C for a week prior to use.

Alsever's solution is an isotonic, anticoagulant blood preservative that permits storage of whole blood at 4C for about 10 weeks.

Ref: Methods in Immunology, A Laboratory Test for Instruction and Research. DH Cambell, et al., WA Benjamin, Inc., 1963. Page 244.

-B-

 

BSA, 3%

Add 3 grams of bovine serum albumin (Fraction V) to 100mL of PBS. Allow to dissolve and store at 4C.

Ref: Antibodies, A Laboratory Manual (1988) Harlow and Lane. Cold Spring Harbor Publications. Page 684.

-C-

 

CaCl2 (for Ca-PO4 transfection)

Calcium chloride dihydrate (CaCl2.2H2O, FW = 147.02)

Prepare a 2M solution (10mL). Measure 2.94 grams calcium chloride and transfer to a 15mL conical tube. Add approximately 8mL deionized water. Vortex to dissolve and bring the final volume to 10mL. Filter-sterilize the solution using a syringe filter (0.22m m). Store at 4C. Keep sterile.

 

Citrate Buffer

16.67mM Na2HPO4 (0.67 grams Na2HPO4.7H2O)

8.33mM Citric Acid (0.26 grams C6H8O7.H2O)

Add ingredients to approximately 100mL deionized water. Adjust the pH to 5.0 and bring the final volume to 150mL.

-D-

     

-E-

 

Ethidium Bromide, 10mg/ml

Add 10 grams ethidium bromide to 100mL deionized water.

Stir on a magnetic stirrer for several hours to ensure that the dye is dissolved. Store in a dark bottle at room temperature.

Ethidium bromide is a DNA intercalating dye and a powerful mutagen. Wear gloves when working with solutions containing this dye.

-F-

     

-G-

 

GBS (Glycine buffered saline)

0.2M Glycine (1.5 grams)

0.1M NaCl (0.58 grams)

Add all ingredients to approximately 75mL deionized water. Adjust the pH to 2.5 using HCl. Adjust the final volume to 100mL.

 

Glucose, 1M

Dissolve 18 grams of glucose in 90mL deionized water. Bring the final volume to 100mL. Filter sterilize using a 0.22m m filter. DO NOT AUTOCLAVE.

-H-

     

-I-

     

-J-

     

-K-

 

KCl, 250mM

Add 1.86 grams KCl to 100mL of deionized water

-L-

 

Laemmli Buffer, 5X

200mM Tris-Cl, pH 6.8 (5mL of 0.5M stock)

400mM b -mercaptoethanol (0.375mL of 14% stock)

8% SDS (0.8 gram)

40% Glycerol (5mL of 100% stock)

0.4% Bromophenol blue (1 milligram)

Add Tris, beta-mercaptoethanol, and glycerol; Mix well. Add SDS. Mix and heat briefly (15-20 seconds) in a microwave to dissolve the SDS. Add the bromophemol blue last. Store at -20C in 1mL aliquots.

 

Luria-Bertani Medium

Per liter:

To 990mL deionized water add

10 grams Bactotryptone

5 grams Bactoyeast Extract

10 grams NaCl

Adjust the pH to 7.0 with 5N NaOH. Sterilize by autoclaving. For LB plates, add 15 grams agar per liter.

Ref: Molecular Cloning, A Laboratory Manual. Eds. Sambrook, et al. (1989)

-M-

 

MgCl2, 2M

Add 19 grams of MgCl2 to 90mL deioized water. Adjust the solution to 100mL. Sterilize by autoclaving.

-N-

     

-O-

     

-P-

 

PBS (Phosphate buffered saline)

100mM NaCl (2.92 grams)

20mM Na2HPO4 (1.42 grams)

5mM KH2PO4 (0.34 grams)

Add all ingredients to approximately 400mL deionized water. Bring the total volume to 500mL. Sterilize by autoclaving.

 

PBS, 1X

NaCl (8 grams)

KCl (0.2 grams)

Na2HPO4.12H2O (2.9 grams)

KH2PO4 (0.2 grams)

Add all ingredients to approximately 800mL deionized water. Adjust the pH to 7.4. Bring the final volume to 1 liter. Autoclave to sterilize and store at 4C.

 

PBS, 10X

NaCl (80 grams)

KCl (2 grams)

Na2HPO4,anhydrous (11.5 grams)

KH2PO4 (2 grams)

Add all ingredients to approximately 800mL deionized water. Adjust the pH to 7.2. Bring the final volume to 1 liter. Add 0.2 grams Thiomersal as a preservative, if desired (cannot be used for tissue culture). Store at 4C. This solution is 0.01M with respect to phosphate and 0.15M with respect to NaCl. Dilute 1:10 prior to use.

Ref: Liddell, JE and A. Cryer. A Practical Guide to Monoclonal Antibodies. John Wiley and Sons. Page 156 and 160 (1991).

 

PCR Buffer

Stock Solutions:

30mM MgCl2

0.2M MOPS, pH 7.75

0.5M KCl

Working Solutions: Add 100m l of each stock solution to a microcentrifuge tube. Bring the final volume to 1000m l.

Working Concentrations:

3mM MgCl2

20mM MOPS

50mM KCl

200m M each dNTP

 

Phosphate Buffer

Stock solutions

Solution A: 0.2M solution of monobasic sodium phosphate (27.8g in 1000ml) (acid)

Solution B: 0.2M solution of dibasic sodium phosphate (53.65g of Na2HPO4.7H2O or 71.7g of Na2HPO4.12H2O in 1000ml) (base)

To prepare a phosphate buffer at a specific pH, use the chart to find the volumes of solution A and B needed, then x ml of A + y ml of B, to make the buffer.

x y pH x y pH

93.5 6.5 5.7 45.0 55.0 6.9

92.0 8.0 5.8 39.0 61.0 7.0

90.0 10.0 5.9 33.0 67.0 7.1

87.7 12.3 6.0 28.0 72.0 7.2

85.0 15.0 6.1 23.0 77.0 7.3

81.5 18.5 6.2 19.0 81.0 7.4

77.5 22.5 6.3 16.0 84.0 7.5

73.5 26.5 6.4 13.0 87.0 7.6

68.5 31.5 6.5 10.5 90.5 7.7

62.5 37.5 6.6 8.5 91.5 7.8

56.5 43.5 6.7 7.0 93.0 7.9

51.0 49.0 6.8 5.3 94.7 8.0

To finely adjust the pH, dilute the required solution to the desired pH with the appropriate stock solution. The stock solution to use depends on the pH desired. Stock solution A will lower the pH, while stock solution B will raise the pH.

 

Polybrene, 10mg/mL

Prepare a stock solution containing 10 milligrams of polybrene per milliliter of PBS. Filter-sterilize using a syringe filter (0.22m m). This is a 1000X solution.

Polybrene Is a polycation that aids in binding of retroviral particles to the cell membrane.

Ref: Practical Molecular Virology. Methods in Molecular Biology, Volume 8. Ed. MKL Collins. Page 31 (1991).

-Q-

     

-R-

 

RBC Lysis Buffer, pH 7.2

Also Tris buffered ammonium chloride

Stock Solutions:

NH4Cl (0.83% weight by volume)

Tris (2.06% weight by volume), pH 7.65

Working Solution:

9 volumes NH4Cl

1 volume Tris-Cl, pH 7.65

Filter sterilize.

Ref: Lymphocytes, A Practical Approach. Ed. GGB Klaus, IRL Press. SV Hunt. Isolation of Lymphocytes and Accessory Cells, Page 32.

-S-

 

Sequencing gel, see acrylamide gel

 
 

SOC

Per liter

To 900mL deionized water, add

20 grams bactotryptone

5 grams bactoyeast

0.5 grams NaCl

Mix and add 10mL of 250mM KCl. Adjust the pH to 7.0 with 5N NaOH. Bring the final volume to 1 liter and sterilize by autoclaving. Before use add 5mL of 2M MgCl2 and 20mL of 1M glucose.

Store SOC in 1mL aliquots at 4C.

 

Solution I, II, and III, see Alkaline Lysis buffers

 

-T-

 

TAE (Busch buffer)

40mM Tris

20mM Sodium acetate

2mM EDTA

Add ingredients to approximately 800mL deionized water. Adjust the pH to 7.5 with glacial acetic acid. Bring the final volume to 1 liter.

For 1 liter of 10X buffer:

Tris (48.25 grams)

Sodium acetate (27.2 grams)

EDTA (7.45 grams)

 

TBE, 10X

121.1 grams Tris

7.4 grams EDTA

55 gram Boric Acid

Dissolve the ingredients in approximately 800mL deionized water; pH the solution to 8.3 with dry boric acid. Filter. Makes 1 liter.

 

TBS (Tris buffered saline), 1X

20mM Tris (2.42 grams)

0.5M NaCl (29.22 grams)

Dissolve the ingredients in approximately 800mL deionized water. Adjust the pH to 7.5 with concentrated HCl (approximately 1.5mL). Adjust the final volume to 1 liter.

 

TBS, 10X

12.1 grams Tris

40 grams NaCl

Dissolve the ingredients in approximately 400mL deionized water. Adjust the pH to 7.6 with approximately 19mL concentrated HCl.

For 4 liters, add 96.8 grams Tris and 320 grams NaCl

 

TE

10mM Tris-Cl, pH 7.4

1mM EDTA, pH 8.0

 

TE, Low

3mM Tris-Cl, pH 7.4

0.2mM EDTA, pH 8.0

Good storage buffer for DNA. EDTA concentration will not inhibit enzymatic reactions. Will inhibit nuclease activity. Will not interfere with subsequent use of DNA (e.g., restriction digests, ligation, transfection, etc.).

 

Tris

Tris (hydroxymethyl)-aminomethane

 

TTBS (Tween plus TBS)

TBS containing 0.2% TWEEN 20 (0.2mL per 100mL TBS)

TWEEN - Polyoxyethylene sorbitan monolaurate

     
     
     
     

-U-

     

-V-

     

-W-

     

-X-

 

X-gal reagent (for eukaryotic staining)

Per milliliter of reagent:

942m L PBS

20m L 200mM K4Fe(CN)6.3H2O

20m L 200mM K3Fe(CN)6

2m L 1M MgCl2

Mix by vortexing and add 16m L of 2% X-gal in dimethyl formamide

-Y-

     

-Z-

     

Vectors : Plasmids and Retroviruses


Plasmids

Plasmids are small circular DNAs (1 kb to more than 200 kb) that are naturally found in many bacteria. Many plasmids encode antibiotic resistance and bacteria that carry such plasmids benefit from expression of these genes. All plasmids replicate independently of the host chromosome, but do require host cell enzymes and factors for replication. 

In the molecular biology laboratory plasmids are used to manipulate DNAs of interest through the process of cloning. These plasmids have been modified to contain strong origins of replication, a selectable marker (e.g., ampicillin resistance gene), cloning sites, and other specialized sequences. The choice of plasmid depends on the future use of the clone. Simple cloning vectors are often used as a vehicle to produce many copies of the DNA of interest for techniques such as sequencing. Expression plasmids contain eukaryotic gene elements (i.e., enhancer, promoter, and poly-Adenylation site) that allow the DNA of interest to be expressed in a eukaryotic cell. 

Plasmids are introduced into bacterial cells through the process of Transformation. Because plasmids express a selectable marker, the cells that take up the plasmid are resistant to a particular antibiotic and have become "transformed". Such bacteria are plated onto semisolid medium containing the antibiotic. The replication of resistant cells creates colonies that consist of many cells that arose from a single plated bacteria containing the plasmid. Therefore all of the cells within the colony are clones. A sample of the colony can be isoloated and grown in liquid culture to obtain high bacterial numbers. The number of plasmid copies per cell can be as high as 100. Since there are millions of cells in the resulting culture, the amount of plasmid DNA has been tremendously amplified.  

The final step is to reisolate the plasmid from the bacterial cells. This is a fairly simple process that uses an alkaline-lysis procedure. The resulting plasmid DNA can then be used in many downstream processes.

Retroviral vectors

The retroviral genome consists of two strands of mRNA packaged within a protein coat (capsid). Upon infecting a host cell, the RNA genome is reverse transcribed into a double-stranded DNA, called a provirus. the provirus is subsequently inserted into the host cell' chromosome where it acts as a normal genetic element. The provirus is characterized by 5' and 3' Long Terminal Repeat (LTR) sequences that flank the virus gene sequences. Generally, the LTRs function in gene expression and insertion into the host cell's chromosome. More specifically, the 5' LTR contains the viral enhancer and promoter sequences as well as the transcriptional start site. The 3' LTR contains the viral poly-Adenylation site. Retroviral transcription results in a single RNA that is alternatively spiced to produce the various viral mRNAs. At minimum the retroviral genome encodes three genes termed gag, pol, and env. Gag encodes the viral capsid proteins, pol the viral enzymes, and env the viral glycoproteins. More complex viruses, such as HIV encode a number of accessory genes. The provirus also encodes a packaging signal that enables the full-length viral genomic RNA to be packaged into virus capsid in preparation for egress from the cell.  

Retroviral vectors are created when plasmids are modified to contain provirus DNA. Such plasmids are further modified by the removal of all viral genes. These "empty" retroviral sequences are customized by the addition of numerous genes and expression cassettes. Depending on the natrure of the vector and the cloning site, a DNA of interest can be expressed from either the viral promoter or internal non-viral promoter. These DNAs can be used as either a plasmid or to generate retroviral particles that express the DNA of interest.

Studies involing DNAs of interest are often contingent on the method of gene delivery to the target cell (tranfection). Transfection methods are categorized as chemical, mechanical, or biological. Chemical methods, such as calcium-phosphate precipitation or lipofectin, take advantage of the formation of large DNA complexes that are taken up by the target cells. The most common mechanical method is electroporation in which plasmid DNA is driven into target cells using a brief electrical pulse. The most common biological delivery methods utilized viral vectors (e.g., retroviral vectors).

Recombinant retroviral particles can be generated by the co-transfection of eukaryotic cells with a retroviral vector and plasmids that express the viral stuctural genes (gag, pol, and env). The recombinant virus particles produced by these cells contain the retroviral genome transcribed from the cloned and modified provirus. These virus particles can be harvested from the producer cells and used to transduce the ultimate target cell. The virus itself is replication incompetent because it cannot express any of the viral structural genes. Transduced target cells can be analyzed for expression of the DNA of interest.