Download Materials and Methods (MMs)

Scientific Reading and Writing in English
Material and Methods
Shu-ying Wang, Ph.D.
Dept. Microbiology and Immunology
Tel: +886-6-2353535 ext. 5634
Fax: +886-6-2082705
Email : [email protected]
The “Materials and Methods (MMs)”
is to address
“what/how you did it”.
Material and Methods
reads like a cookbook
Need to provide sufficient information so that
the readers can exactly reproduce the
experiment
Material and methods
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Subjects
Sample preparation techniques
Sample origins
Data collection protocol
Data analysis techniques
Any computer programs used
Description of equipment and its use
Spacing
 …..previously described (30)
 2 mM, 2 h (2 hr), 2 min, 2 sec, 2 pg/ml, 1 ml, pH 6.8,
 5%, 4°C, -80°C, ~ 40,
 171 mm, 4 Ǻ
 a = b = C = 152 mm, 0.15 x 0.15 x 0.2 mm
 1-368, 22-mer DNA
What is “MMs” and what should be in the “MMs” section?
“MMs”. The “MMs” section should include sufficient technical
information to allow the experiments to be repeated. When
centrifugation conditions are critical, give enough information to
enable another investigator to repeat the procedure: make of
centrifuge, model of rotor, temperature, time at maximum speed,
and centrifugal force ( X g rather than revolutions per minute). For
commonly used materials and methods (e.g., media and protein
concentration determinations), a simple reference is sufficient.
-continued
If several alternative methods are commonly used, it
is helpful to identify the method briefly as well as to
cite the reference. For example, it is preferable to
state ‘‘cells were broken by ultrasonic treatment as
previously described (9)’’ rather than to state ‘‘cells
were broken as previously described (9).’’ The
reader should be allowed to assess the method
without constant reference to previous publications.
-continued
Describe new methods completely and give sources of unusual
chemicals, equipment, or microbial strains. When large numbers of
microbial strains or mutants are used in a study, include tables
identifying the sources and properties of the strains, mutants,
bacteriophages, plasmids, etc. A method, strain, etc., used in only
one of several experiments reported in the paper may be described
in the Results section or very briefly (one or two sentences)
in a table footnote or figure legend.
Example:
Cells, viruses, and reagents. The B-cell line Raji was
maintained in RPMI medium containing 10% fetal bovine serum
according to American Type Culture Collection instructions. DV2
strains PL046 and M16681 were propagated in C6/36 mosquito
cells and titrated on BHK cells as previously described (18).
Human DV3 immune serum was obtained with consent from an
infected patient. The titer of this serum was 1:12,000 against DV3
and 1:3,200 against DV2 strain PL046, as determined by
measuring 50% plaque reduction in a neutralization assay using
BHK cell monolayers (14). Control serum was collected from a
healthy blood donor without DV-specific antibodies in serum as
determined by an enzyme-linked immunosorbent assay (ELISA)
as previously described (19).
-continued
A monoclonal antibody (MAb) against the viral envelope protein
was obtained from Endogen (Woburn, Mass.), and a MAb against
the viral core protein with high specificity was purified from
supernatant of hybridoma cells as previously described (40).
Flow cytometry. Infected B cells were washed with phosphatebuffered saline (PBS) twice, fixed, permeabilized, washed, and
incubated with the IgG fraction of NS1 hyperimmune serum or
normal mouse serum. Cells were then washed and incubated with
fluorescein isothiocyanate-conjugated goat anti-mouse IgG
(Endogen). After incubation, cells were washed twice with PBS,
resuspended in PBS, and then analyzed with a FACScan flow
cytometer (Becton Dickinson Immunocytometry Systems, San
Jose, Calif.) as previously described.
-continued
Cytokine measurement. Cytokines were measured by
ELISA kits according to the manufacturer’s protocols. The
detection limits for the cytokines (Endogen) were as follows:
TNF-a, 16 pg/ml; IL-6, 11 pg/ml.
Statistical analyses. In vitro data were analyzed by Student’s
t test. Viral loads in tissues were analyzed by Mann-Whitney
U test. Kaplan-Meier survival curves were analyzed by logrank test. All P values are for two-tailed significance tests.
•Use subheadings to organize “MMs”.
•Previous papers in the laboratory are good examples.
•Chronological order of “MMs” (Which should come first?)
Match the chronological order of “MMs” with that of the “Results”.
However, related methods should be described together.
The recombinant cED III was expressed in E. coli
BL21-codonPlus(DE3)-RIL (Stratagene, La Jolla, CA). After
isopropylthiogalactoside (IPTG) induction, the recombinant
cED III was purified with immobilized metal affinity chromatography
(IMAC) columns. The eluent from the IMAC column
was then polished by using an anion exchange column (Q
sepharose fast flow; GE) after dialysis against Q buffer (20 mM
Tris-Cl, 1mMEDTA, pH 8.0).
Recombinant native FlhDC protein complex was
expressed using methods described elsewhere1. Briefly,
the protein was expressed in E. coli strain BL21(DE3) (B Fdcm ompT) carrying the plasmid pSW28 bearing the
tandem genes of the flhDC operon (pET24a-flhDC), and
was purified by affinity chromatography (HiTrap heparin,
Pharmacia) followed by size-exclusion gel-filtration
(Superdex 200, Pharmacia).
Homework:
1. Practice to write your own “Material and Methods”
section