MBIO 4610
Molecular Genetics of Eukaryotes Lab

MBIO 4610 Teaching Assistants:
April Gislason, Jennifer Tanner, Tanya Pribytkova


All lab reports must have a completed Honesty Declaration attached (pdf): Group Lab Assignment or Individual Lab Assignment.

Lab starts dates: Monday September 12 (201 Buller) and Wednesday September 14 (201 and 204 Buller). Lab manuals are handed out in class the first week of lectures. All essential lab related information is given in the lab manual.

Lab Exam Outline

Lab report marks

Lab exam mark and final lab marks

CHANGE for MONDAY LAB: Since Monday Oct 10, 2011 is Thanksgiving the lab will be held Wednesday Oct 12, 2011 at 4 pm in room 201.

Lab Start Information
·Introduction to lab. Discussion of student pre-lab set up required for next week’s lab.
·Written primer design quiz (written as a group). Open book, includes lab manual and any additional information you want to bring related to the orientation of primers. Come prepared.

·Practical Pipetmen operation quiz (individually) using P200 (20 µl & 180 µl). See lab manual appendix for Pipetman operation. You must be checked off before leaving the lab (no marks allotted but marks subtracted if not completed).
·Come to the first lab with the written Pipetman questions completed by each student (see lab manual) or linked below. Marked in-lab by the instructor. Mark is part of the your Primer Quiz mark.

Lab Reports and Assignments:

Read lab report presentation section in general instructions at the beginning of your lab manual before doing your lab report. Lab reports must typed. This includes calculations. 10% of the mark subtracted for portion of report not typed.

Pipetman Written Quiz (Save and open in Word to enter required information).

Lab 1. PCR amplication of human polymorphic mitochondrial DNA

lab 1 report format (Word). Save word document before entering requested information.

Lab 1 report spreadsheet (Excel). Save spreadsheet and open in Excel before entering requested information. Correction to lab manual Excel DNA size (bp) Determination Standard Curve information - should say Select Insert pull down menu. Select Scatter. Next select scatter with markers not connected by lines. An empty chart box appears in spreadsheet.

Student HaeIII digested human mtDNA agarose gel results (posted when available - see below). jpg files are large to increase resolution, just copy paste into Word document and resize to fit.
Comments:
(1) If you do not have any results for your DNA sample, borrow another student's DNA result(s) and reference for lab report write up. Remember number of DNA samples analyzed must equal the number of students in the group (if handing in report as a group).
(2) If bands are way fainter than the three main bands (most common pattern), do not consider when analyzing data.
Monday Student Sign-up Sheets (pdf):1, 2, 3, 4
Monday Student Gel Photographs (jpg):gel 1, gel 2, gel 3, gel 4
Wednesday Student Sign-up Sheets (pdf):1, 2, 3, 4, 5, 6, 7, 8
Wednesday Student Gel Photographs (jpg):gel 1, gel 2, gel 3, gel 4, gel 5, gel 6, gel 7, gel 8

Lab 2 Lab 2 Ty insertion Mutagenesis and Analysis in Saccharomyces cerevisiae

Lab 2 Respiratory Gene Mutant Selection Group DATA SHEET

Lab 2 report format (Word). Save word document before entering requested information.

Lab 2 report spreadsheet (Excel - class data now available). Select tab at bottom of worksheet to select the appropriate worksheet.Group Southern Blot question has been changed from what is stated in lab manual lab 2 report for #4. Follow information in text box, Excel tab, Group Southern Blot worksheet.

To fit gel and blot side by side in figure: After inserting picture of gel - format is highlighted, select text wrapping square then insert blot. If not side by side resize picture(s) to fit side by side.
Student Sign-up Gel Sheets (pdf):Monday 1, 2, 3, 4; Wednesday 1, 2, 3, 4, 5, 6, 7, 8
Student Southern Gel Photographs (jpg):Monday gel 1, gel 2, gel 3, gel 4; Wednesday gel 1, gel 2, gel 3, gel 4, gel 5, gel 6, gel 7, gel 8.
Information about agarose gels: Printout of gels may not show all plasmid bands, this is okay - still number relevant plasmid bands by estimating position from jpeg picture on lab website. Remember you cannot mark on gel lane. Make sure your plasmid, pGALTyhis3-AI, on the agarose gel is Pst I digested (should be 5 bands, 4 obvious and smaller 0.75 kb fragment unlikely visible. If not Pst I digested, use another group's data and reference. Plasmid should have obvious bands and genomic DNA should be a smear with possibly some multiple bands. Remember any lighter band above a darker band is a partial digest or bands do not decrease in intensity relative to decreasing bp size or more bands than expected especially higher bp. If a blurry band occurs at the running front, it is most likely degraded DNA.

Student Southern Gel Blots (jpg): Monday blot 1, blot 2, blot 3, blot 4; Wednesday blot 1, blot 2, blot 3, blot 4, blot 5, blot 6, blot 7, blot 8. Read information about each blot below.
Information about the blots: Must use corresponding agarose gel and blot for lab report write-up. pGALTyhis3-AI hybridization worked for many groups, expect two bands, partial digest has more bands - need to use another group's data and reference.
Monday blot 3 1st lane only partial digest of pGALTyhis3-AI; dot between lanes points out location of yeast DNA band.
Monday Blot 2 bands were too light to be scanned to jpeg, although present for plasmid.
Monday blots 1 & 4: to permit use of your own gel/blot in report a genomic yeast DNA band has been inserted.
Wednesday blots 1, 3, 4, 6, 7 & 8 has a dot by genominc yeast DNA bands to indicate location of bands as much lighter than plasmid bands. Blots 6 & 8 genomic bands on original blot are very light and barely show up on jpeg file - assume band by dot.
Wednesday blot 4 was not inverted during Southern blot so order of group and loading is reversed; 36, 35, 34 Y2 Y1 P.
Wednesday blots 2, 5 & 7 has an inserted yeast DNA band to permit use of your own gel/blot in report.

Lab 2 Part V. Theoretical genetic database search to identify gene disrupted by Ty element
Assignment (Save word document, open in Word to record requested information.) Changes to lab manual relevant to groups 41-47....group sequence assignment should read 4/41, 5/42, 6/43, 7/44, 8/45, 9/46 and 10/47.

References and Information:
Some references are available on the internet. One option is to use UM Goggle Scholar. Some are also directly linked to downloaded pdf file. Some articles are only available in reference binder on reserve in th Science and Technology Library.

Lab 1. PCR amplication of human polymorphic mitochondrial DNA
QIAGEN DNeasyTissue (also yeast) Handbook
Ginther, C., Issel-Tarver L., King M-C. 1992. Identifying individuals by sequencing mitochondrial DNA from teeth. Nature Genetics 2:135-138.

Lab 2 Lab 2 Ty insertion Mutagenesis and Analysis in Saccharomyces cerevisiae
Saccharomyces cerevisiae gene nomenclature

Databases:
Saccharomyces Genone Database
BLAST the human genome

Curcio, J. M., Garfinkel, D. J. 1991. Single-step selection for Ty1 element retrotransposition. Proc. Natl. Acad. Sci. USA 88: 936-940.

Faculty of Science Cheating, Plagerism,etc

Frequently information is available as a pdf file requiring Adobe Acrobat Reader. Adobe abcrobat reader is available at UM Software ExpressUM Software Express.

Lab Skill Sheet- available for students to use for job applications, just check off the highlighted boxes for the lab course(s) you have taken.
pdf or excel (prior to Fall 2007)
pdf or excel (starting Fall 2007)

email le_cameron@umanitoba.ca
Emails: subject must contain course number and subject, eg. 4610 lab 1 report. If no subject given, email is deleted. Emails replies occur only during working hours. Email must include student name.
phone 474-6542
Department of Microbiology
Buller Bldg
Winnipeg, MB
R3T 2N2