Powerpoint template for scientific poster

1
Advanced Molecular Techniques (Bio. 205) a
Course Overview. Fall 2004 Bio. 205 students and
Dr. Steven White Department of Biology, San
Jose State University, San Jose CA 19192
1 2 3 4 5 6 7
Introduction Students in molecular biology must
gain practical experience in a variety of modern
laboratory techniques if they are to have any
real understanding of how experimental designs
are developed and carried out, if they are to
make effective use of their time in the research
lab, if they are to be competitive in todays job
market etc. Bio. 205 was developed to address
some of that need for practical experience, with
experiments and techniques presented in a logical
progression and linked format (with one
experimental result used as input to another) to
more closely approximate industrial or graduate
thesis research experience. The technique
summaries presented here are meant to provide the
reader with an experimental context (there is
simply insufficient room to include actual
methodological detail), but all data is student
group generated and representative of all group
results. Note that parallel group research
projects were also performed off hours as an
additional course requirement for Bio.205, but
results there will be the subject of another
poster.
Fig. 14. Example of in vitro transcription
reaction products run out on a 1 native agarose
gel just prior to use with Ambions
photoactivatable psloren- biotinylation system.
The biotinylated probe was then used in a
Northern.
Note that the robust production of transcript in
lane 3 is due to the use of Ambions MEGAscript
in vitro transcription system, which is optimized
for use with template designed to produce
transcripts of suggests that short RNAs are more efficiently
synthesized using this modified transcription
system.
10 1 0.1 0.01
Fig. 7. Example of Southern hybridization
results. The biotinylated Hind III Lambda
fragment was used to probe dilutions of a Lambda
DNA digest run in a background of calf thymus
DNA, which allowed highly quantitative analysis
of the sensitivity of this method.
procedure using Novagen Inc reagents) linearized,
blunt-ended pST Blue-1 supplied by Novagen.
Following ligation, the products were employed in
a chemical transformation according to protocols
supplied by Promega or Novagen, respectively.
Transformants were plated on LA/Amp/IPTG/XGal
plates for blue/white screening, and numerous
white colonies subsequently grown up for plasmid
isolation. The plasmids so derived were screened
by PCR using T7 and SP6 priimers to identify
those with appropriately sized inserts. Cycle
(DNA) Sequencing Recombinant plasmids resulting
from the TA and blunt-end cloning experiments
were then employed as templates in either a Big
Dye or d-Rhodamine cycle sequencing protocol.
The chain extension products were precipitated,
washed, lypholyzed and resuspended following
standard protocols (see ABI Prism 310 sequencing
manual) prior to capillary electrophoresis (1,
2). cDNA production for cDNA library
construction Mouse mRNA was used in first and
second strand cDNA synthesis using Novagens
OrientExpress cDNA synthesis kit following the
manufacturers protocols (1, 2). Construction of
a cDNA library in Lambda Phage The resulting
double stranded cDNAs were end-polished,
methylated in vitro, ligated to linkers,
restriction digested and and the linker digestion
products and free linkers removed using molecular
filtration spin columns. The ligation-ready
cDNAs were then ligated to Novagens LambdaScreen
pre-digested Lambda arms and then packed in
vitro using Novagens packaging reagent system.
Packaged recombinant Lambda phage were then used
to infect ER1647 E. coli host cells, plated in
top agar over agarose using standard methods
(Maniatis et al), and the resulting viral plaques
counted and ultimately harvested (using the
flood plate technique) and stored at -70 oC for
use by subsequent classes (1, 2).
Lane 1 100bp ladder, Lane 5 Low mass
InVitrogen DNA ladder. Other lanes are various
student group results.
(A)
(B)
(C)
1 2 3 4 5 6 7
Fig.15. Examples of group Northern blots. A
typical denaturing agarose gel run (A) and the
corresponding X-ray film following hybridization,
washing and detection (B). Film in (C) shows
another blot. Numbers below A indicate amount of
total RNA (lanes1-6) or mRNA (lane 7) loaded in
each lane.
Numbers above each lane correspond to total
amount of Lambda DNA loaded (in nanograms), while
numbers below are target DNA mass detected (in
picograms).
470 47 4.7 0.47
Fig. 8. Results of 2-dimensional protein gel
electrophoresis using two different protein
extraction protocols (IEF gel pH gradient of pH
4-7, SDS-PAGE gradient of 8-16 polyacrylamide).
Many proteins isolated by the second protocol
clearly are underrepresented.
Mouse Tissue
Linearized transcription vector containing insert
Total RNA isolation
Overview of Materials and methods All reagents
were prepared by staff, obtained commercially or
donated. We especially acknowledge both the
Ambion and Novagen companies for their generous
support. Plasmid Isolation and Characterization
Plasmids were isolated using both in house
prepared reagents for a standard alkaline lysis
protocol (1, 2) and commerically obtained Qiagen
SpinPreps (Qiagen Inc.) to allow comparison of
average yield, degree of contamination (as
measured by OD260/280 ratio, 210nm-300nm
absorption spectrum analysis and agarose gel
electrophoresis AgGE) and functionality (as
measured by capacity for restriction digestion
and religation). Parallel DNA samples were
spiked with contaminants (ie chaotropes,
solvents, protein, salts etc) prior to
spectrophotometry, AgGE or functional tests for
purposes of comparison. Additionally, DNAs were
sometimes further purified and concentrated using
spin ultrafiltration via Amicon-30 spin filters
(Amicon Inc). Restriction Digests Agarose Gel
Electrophoresis (AgGE) All restrictions were
performed according to standard protocols (1, 2),
using reagent grade water and commercially
obtained enzymes and 10X buffers (Promega Inc).
Native AgGE runs (using TAE, TBE or MOPS buffers)
were performed using in house prepared reagents
(1, 2). Denaturing (formamide/formaldehyde MOPS)
AgGE runs for RNA utilized reagents supplied by
Ambion Inc, and were performed using the Ambions
protocols (essentially those described in
references 1 and 2). DNA was isolated from
excised agarose gel bands using a QIAquick kit
(Qiagen Inc) based protocol. Genomic DNA
isolation and PCR E.coli genomic DNA was
isolated using both in house prepared reagents
and a standard CTAB protocol (2) and via a Gentra
DNA isolation kit. The DNAs were analyzed by
AgGE, then used in a PCR reaction (3) to amplify
a 730 bp fragment of the Trp A gene. PCR product
was then analyzed by AgGE against mass and size
standards. Originally we intended to run an
second Southern using this TrpA gene fragment as
a probe, but scheduling problems prevented
it. DNA labeling for use in Southern
Hybridization Probe DNA (a 2.3 kbp Hiind III
fragment from Lambda phage) was labeled using the
Phototope system kit (New England Biolabs Inc)
via a random priming protocol (1, 2) using both
biotinylated primers and a biotinylated
dNTP. Southern Hybridization Varying amounts of
Hind III digested Lambda genomic DNA (target DNA)
diluted in irrelevant calf thymus DNA (to add
sequence complexity to the target population)
were electrophoresed on a native agarose gel and
transferred to nylon via capillary blotting
according to standard protocols (Maniatis et al).
Hybridization (probe at 20 ng/ml) was carried
out overnight at 68 oC following the NEB protocol
with standard hyb solution or 42 oC with
Ultrahyb solution, and hybrids detected using
the NEB Phototope Detection system following the
NEB protocol. X-ray films were developed using
Kodak GBX developer fixer solutions following
standard Kodak protocols (1, 2, 5). Protein
isolation from E. coli using various extraction
protocols. Bacterial proteins were isolated using
one of 3 different extraction protocols in which
the nature and/or concentration of the non-ionic
detergent were varied. Protein content of each
fraction was then determined using BioRads RC DC
protein assay kit. 2D-PAGE (IEF and SDS-PAGE) of
extracted bacterial proteins. Samples were
diluted in BioRads IEF sample loading buffer and
applied to pH 4-7 Immobiline IEF strips and
subjected to isoelectric focusing on a BioRad
Protean IEF unit following their recommended
protocol. The focused IEF strips were then
soaked in SDS-PAGE sample buffer and applied to a
Criterion 10.5 to 14 gradient SDS-PAGE gel,
electrophoresed, and stained using Ruby Red
following manufacturers protocols
(6). Isolation of Mouse Total RNA
Total RNA was isolated from mouse liver and
heart tissue using both the TRI reagent (Sigma
Inc) and TotallyRNA prep (Ambion Inc) protocols
following manufacturers instructions (1, 2, 4).
The RNAs were analyzed for yield, purity, size
range and degradation via spectrophotometry and
AgGE. Total RNAs were then stored in DEPC-treated
water at -70 oC. Isolation of Mouse mRNA mRNAs
were isolated from total RNA via Ambions
Poly(A) Purist kit and protocol (4). The
resulting mRNA preps were then analyzed via both
spectrphotometry and AgGE. In Vitro
Transcription A linearized plasmid containing a
285 bp fragment of the mouse actin gene was used
in a run off in vitro transcription protocol
utilizing either Ambions MAXIscript or
MEGAscript In Vitro RNA synthesis kit. The
transcripts were then analyzed by AgGE, and
purified for RNA labeling (1, 2, 4). RNA
labeling for use in Northern Hybridization RNAs
to be employed as hybridization probes were
post-synthetically biotinylated using Ambions
BrightStar Psoralen-Biotin labeling
system. Northern Hybridization Mouse total and
mRNA samples were run out on denaturing gels and
blotted by capillary transferto nylon membranes
following standard protocols (Maniatis et al).
Biotinylated probe (appeox. 0.1 ng/ml) was
diluted in Ambions Ultrahyb solution, hybridized
at 42 oC overnight, and the blot then washed and
hybrids detected using Ambions NorthernMax and
BrightStar Detection systems (1, 2, 4,
5). RT-PCR Mouse total RNA and class-designed
primer sets were employed to specifically amplify
a mouse Nkx2.5 gene fragment or actin gene
fragment using Qiagens One-Step RT-PCR system,
with the products subsequent used in both blunt
end and TA cloning efforts. Following RT-PCR, the
products were analyzed by AgGE, then cleaned or
band purified prior to their use in ligation (1,
2, 3). TA and Blunt end Cloning The purified
DNA fragments generated by RT-PCR were next
ligated into both a linearized T-ended vector
(pGEM T-Easy from Promega Inc) or (following an
end polishing
Fig. 2. Flow chart summarizing experimental
design for the second portion of the course.
In Vitro Transcription and cleanup
mRNA isolation
RT-PCR and cleanup
AgGE and band isolation
Denaturing AgGE and Capillary Blot
Riboprobe Labeling
TA and Blunt end Cloning
18 9 4.5 2.25 1.12 0.62 1
Plasmid isolation
Northern Hybridization and Detection
cDNA Library construction in Lambda Phage
Cycle DNA Sequencing
Fig. 16. Photographs at right show representative
plates of recombinant Lambda phage, as viral
plaques on a lawn of host E. coli. Mouse mRNA
was used to create cDNA libraries cloned into
Novagens LambdaScreen vector.
Results
Fig. 3. Example of (A) absorbance scans of pure
DNA vs (B) DNA in the presence of a contaminant
(in this case, 5 v/v guanidine hydrochloride),
(C) spot gel showing DNA dilution series, and
(D) short run gel showing DNA dilution series
(both used to quickly estimate DNA concentrations
in small volumes of unknowns).
1 2 3 4 5 6
7
Fig. 9. Examples of mouse total RNA isolated
using Ambion RNAeasy (lanes2-4) vs total RNA
isolated using TRI reagent (lanes 5-7). In both
cases the RNA appears intact (sharp 28s and 18s
bands). Note, however, that the RNA isolated
using TRI reagent was often significantly
contaminated with genomic DNA, and thus
frequently required extensive DNase digestion
prior to use in RT-PCR or cDNA library
construction. Gel on right is RNAeasy prep only
Conclusions Results shown here provide some
indication of the technical experience gained by
the Bio205 students. Other experience, which is
difficult to present in this condensed format but
is nonetheless important, involved their skills
development in overall project design, individual
experimental design, technique problem diagnosis
and treatment, database mining, literature
analysis and goals assessment. In addition to the
accompanying instructor lectures (in which
underlying theory and technical aspects are
discussed), students take two 3 hour written
exams, carry out a supplementary research project
(beyond the work presented here), create and turn
in (for graded evaluation) a research notebook
detailing all their work in the course and
discussing their results, and give a 15 min
Powerpoint presentation (ASM or ASCB format) on
one of various instructor-designated research
topics. Despite the rigor of the course, students
unanimously agreed the experience was extremely
valuable.
(A)
(B)
(D)
(C)
Note that the OD260/280 ratios in A and B are
essentially identical (1.75 vs 1.69) while their
absorption spectra clearly are not, illustrating
the importance of spectrum analysis in assessment
of DNA purity.
Fig. 4. Examples of 1 agarose TAE gel runs. (A)
Dilutions of purified plasmid (uncut). (B)
Plasmid restricted with Hind III to liberate
insert. (C) Gel following insert band incision.
(D) Gel showing purified inserts ready for
biotinylation.
Fig. 11. Example of RT-PCR products generated
using total RNA and a thermal gradient cycler to
identify the optimal primer annealing temperature
for amplifying a mouse Nkx2.5 gene fragment.
Products anlyzed on a 2 native agarose gel.
1 2 3 4 5 6
Fig. 10. Example of purified mRNA (isolated using
Ambions MicroPolyA Pure kit, starting from
total RNA), prior to use in Northern blot
experiments and cDNA library construction.
Bacterial cells
Plasmid isolation and purity analysis
Genomic DNA isolation
Protein extraction and sample prepartion
Fig. 1. Flow chart summarizing experimental
design for the first portion of the course.
(B)
(C)
(D)
Restriction digestion and AgGE
(A)
Isoelectric focusing
Band purification
PCR (at various annealing temps and/or template
amounts)
Random priming-based DNA labeling and probe
purification
SDS-PAGE (2-Dimensional gel electrophoresis)
Literature cited 1. Sambrook, J., Fritsch, E.F.,
and T. Maniatis. 1989. Molecular Cloning, A
Laboratory Manual. Cold Springs Harbor Press. 2.
Ausubel, F. et al. 1997. Short Protocols in
Molecular Biology. John Wiley Sons. 3. Newton,
C.R., and A. Graham. 2000. PCR (Second Edition).
Springer Pub. Co. 4. Farrell, R. 1993. RNA
Methodologies A Laboratory Guide for Isolation
and Characterization. Academic Press. 5. Hames,
B.D., and S.J. Higgins. 1985. Nucleic Acid
Hybridization A Practical Approach. IRL
Press. 6. Hames, B.D., and D. Rickwood. 1990.
Gel Electrophoresis of Proteins A Practical
Approach. IRL Press.
Southern blot, hybridization and detection
AgGE
Fig. 12. Example of RT-PCR products generated
using mouse total mouse RNA and actin primers.
Optimal annealing temp 60 oC, lane 6
Fig.6. Examples of preliminary Southern
hybridization experiments using (A) standard hyb
buffer (Maniatis et al) vs (B) Ambion Ultrahyb
buffer.
Fig. 5. Example of 2 agarose gel run of TrpA PCR
product. This fragment will be cloned for use in
a later Bio205 class.
(A)
Acknowledgments We thank D. Leeve and L. Goff for
technical assistance, and all those at both
Ambion and Novagen Corporations for their
generous support.
Fig. 13. Examples of blunt end cloning vectors
(A) and TA cloning vectors (B) used in the
construction of recombinant plasmids containing
mouse Nkx2.5 or mouse actin RT-PCR fragments.
Both fragments were successfully cloned in both
vectors. Positive actin clones, identified
using a T7/SP6 primer-based PCR technique, are
shown in (C) and (D).
(A)
(B)
For further information It is important to note
that Bio. 205 is not atypical, but is in fact
representative of upper division and graduate
level lab courses in the Dept. of Biology as
SJSU. See, for example Bio. 205T, Bio. 233, Bio.
234, Bio. 227, Bio. 135/135L, Bio. 124/125,
Micro. 141/141L etc. etc. Please visit the SJSU
web site, call the SJSU Dept. of Biology or
contact Dr. White at sjwhite_at_email.sjsu.edu for
more details.
(B)
The Ultrahyb buffer gave a much stronger signal
for all groups.
(C)
(D)