学术会议海报模板10academicconferencepostermodel

《学术会议海报模板10academicconferencepostermodel》由会员分享，可在线阅读，更多相关《学术会议海报模板10academicconferencepostermodel（1页珍藏版）》请在装配图网上搜索。

1、Acknowledgments We thank D. Leeve and L. Goff for technical assistance, and all those at both Ambion and Novagen Corporations for their generous support.,Introduction Students in molecular biology must gain practical experience in a variety of modern laboratory techniques if they are to have any rea

2、l 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 t

3、echniques 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

4、” (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 th

5、ere will be the subject of another poster.,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 isolat

6、ed 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 el

7、ectrophoresis 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,

8、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 obtai

9、ned 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

10、(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 Gen

11、tra 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

12、 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 d

13、NTP. 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 stan

14、dard 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

15、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-io

16、nic 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 is

17、oelectric 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). Iso

18、lation 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 spectrophotom

19、etry 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 linearize

20、d 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 u

21、se 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 memb

22、ranes 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 to

23、tal 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, t

24、hen 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”,Results,Conclusions Results show

25、n 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 prob

26、lem 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

27、 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

28、course, students unanimously agreed the experience was extremely valuable.,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,Literature cited 1. Sambrook, J., Fritsch, E.F.

29、, 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 Lab

30、oratory 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.,Fig. 1. Flow chart summarizi

31、ng experimental design for the first portion of the course.,Fig. 5. Example of 2% agarose gel run of TrpA PCR product. This fragment will be cloned for use in a later Bio205 class.,Fig. 7. Example of Southern hybridization results. The biotinylated Hind III Lambda fragment was used to probe dilution

32、s of a Lambda DNA digest run in a background of calf thymus DNA, which allowed highly quantitative analysis of the sensitivity of this method.,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

33、 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 sjwhiteemail.sjsu.edu for more details.,procedure using Novagen In

34、c 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 nume

35、rous 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 e

36、mployed 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

37、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

38、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 r

39、eagent 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

40、use by subsequent classes (1, 2).,Fig. 2. Flow chart summarizing experimental design for the second portion of the course.,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 diluti

41、on series, and (D) “short run” gel showing DNA dilution series (both used to quickly estimate DNA concentrations in small volumes of unknowns).,(A),(B),(C),(D),Fig. 4. Examples of 1% agarose TAE gel runs. (A) Dilutions of purified plasmid (uncut). (B) Plasmid restricted with Hind III to liberate ins

42、ert. (C) Gel following insert band incision. (D) Gel showing purified inserts ready for biotinylation.,(A),(B),(C),(D),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% polyacrylami

43、de). Many proteins isolated by the second protocol clearly are underrepresented.,Fig.6. Examples of preliminary Southern hybridization experiments using (A) standard hyb buffer (Maniatis et al) vs (B) Ambion Ultrahyb buffer.,The Ultrahyb buffer gave a much stronger signal for all groups.,(A),(B),Fig

44、. 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,

45、 and thus frequently required extensive DNase digestion prior to use in RT-PCR or cDNA library construction. Gel on right is RNAeasy prep only,1 2 3 4 5 6 7,Fig. 10. Example of purified mRNA (isolated using Ambions MicroPolyA Pure kit, starting from total RNA), prior to use in Northern blot experime

46、nts and cDNA library construction.,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,Optimal annealing

47、temp = 60 oC, lane 6,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.,Fig. 12. Example of RT-PCR products generated using

48、mouse total mouse RNA and actin primers.,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 c

49、lones, identified using a T7/SP6 primer-based PCR technique, are shown in (C) and (D).,(A),(B),(C),(D),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 L

50、ambdaScreen vector.,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

51、in each lane.,(A),(B),(C),18 9 4.5 2.25 1.12 0.62 1,1 2 3 4 5 6 7,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.,Note that the robust prod

52、uction 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 about 350 bases. This result suggests that short RNAs are more efficiently synthesized using this modified transcription s

53、ystem.,Lane 1= 100bp ladder, Lane 5= Low mass InVitrogen DNA ladder. Other lanes are various student group results.,1 2 3 4 5 6 7,470 47 4.7 0.47,Numbers above each lane correspond to total amount of Lambda DNA loaded (in nanograms), while numbers below are target DNA mass detected (in picograms).,1

54、0 1 0.1 0.01,Mouse Tissue,Total RNA isolation,RT-PCR and cleanup,AgGE and band isolation,TA and Blunt end Cloning,Plasmid isolation,Cycle DNA Sequencing,mRNA isolation,Linearized transcription vector containing insert,In Vitro Transcription and cleanup,Denaturing AgGE and Capillary Blot,Northern Hyb

55、ridization and Detection,Riboprobe Labeling,cDNA Library construction in Lambda Phage,Bacterial cells,Plasmid isolation and purity analysis,Restriction digestion and AgGE,Band purification,Random priming-based DNA labeling and probe purification,Southern blot, hybridization and detection,Protein extraction and sample prepartion,Isoelectric focusing,SDS-PAGE (2-Dimensional gel electrophoresis),Genomic DNA isolation,PCR (at various annealing temps and/or template amounts),AgGE,