Required Reading. Functional Genomics Research Stream. Pay Attention III III

Transcription

1 Required Reading Functional Genomics Research Stream Research Meeting: March 1, 2011 PCR Mediated Gene Deletion, Transformation of Yeast Screening for Gene Deletions by PCR Genomics Research Agenda Pay Attention IV Enzymatic Assays III Molecular Preparations Aseptic Cell Culture Safety and Basic Operations III II I

2 A Gene is Deleted is replaced with How was the gene deleted? kanmx4 by transformation & homologous recombination s in LB Media s in LB+kanamycin Media

3 s in LB+ampicillin Media Antibiotic Questions How does ampicillin work? How does kanamycin work? How did the cells with the plasmid survive? AmpR polypeptide coding sequence KanR polypeptide coding sequence

4 s in LB+kanamycin Media s in LB+ampicillin Media Critical Question This was a selection. We wish to delete from the yeast genome. This will happen in a process where a very small fraction of cells will actually have the gene deleted. How can we design the deletion process to use the process of selection to select for yeast cells that have had deleted?

5 Answer We will delete. We will replace it with kanmx4. We will select with the eukaryotic version of kanamycin (geneticin, G418). We will analyze the genomic DNA of cells that survive in order to prove is indeed gone and kanmx4 is present (at the right spot in the genome). A Gene is Deleted is replaced with kanmx4 by transformation & homologous recombination Gene Deletion 1. Purify DNA plasmid containing kanmx4 fragment by growing E. coli and mini-prepping. 2. Use PCR to build DNA fragment you wish to incorporate into the genome. This fragment must contain a selectable marker. The process. 3. Transform fragment into live cells. 4. Allow cells to incubate with fragment - homologous recombination occurs. 5. Plate cells on selective mediate (YPD+G418). 6. Allow cells to grow for 48 to 72 hours. 7. Select and label colonies, grow overnight culture for each colony selected. 8. Prepare genomic DNA for each overnight, quantitate. 9. Run PCR reactions to interrogate genomic DNA for existence of original gene, incorporation of kanmx4 fragment. 10. Analyze PCR reactions by gel electrophoresis.

6 1. Purify DNA plasmid containing kanmx4 fragment by growing E. coli and miniprepping. 2. Use PCR to build DNA fragment you wish to incorporate into the genome. This fragment must contain a selectable marker. Round 1 PCR UPTAG primer produces DOWNTAG primer GenElute Plasmid Miniprep Kit s with plasmid (cell culture, media is LB+kanamycin) 2. Use PCR to build DNA fragment you wish to incorporate into the genome. This fragment must contain a selectable marker. Round 2 PCR UP_XX primer DOWN_XX primer produces

7 3. Transform fragment into live cells. yeast cell 4. Allow cells to incubate with fragment - homologous recombination occurs. CHR III CHR III AAAAAAAAAAAAAAA AAAAAAAAAAAAAAA AAAAAAAAAAAAAAA AAAAAAAAAAAAAAA yeast cell yeast cell AAAAAAAAAAAAAAA

8 5. Plate cells on selective mediate (YPD+G418). YPD + G418 plate 6. Allow cells to grow for 48 to 72 hours. yeast cell AAAAAAAAA AAAAAAAAA AAAAAAAAA AAAAAAAAA AAAAAAAAA cells that survive G418 treatment produce a colony Selective Media:YPD+G Select and label colonies, grow overnight culture for each colony selected. 8. Prepare genomic DNA for each overnight, quantitate. Transformed Cells on YPD Transformed Cells on YPD+G418

9 YPD+G Run PCR reactions to interrogate genomic DNA for existence of original gene, incorporation of kanmx4 fragment. required gene Y Overnight Cultures pb pc pd ~500 bp ~500 bp +F ~200 bp +R PCR product kanmx4 required gene Y Genomic DNA Preparations pkanb pkanc pd ~500 bp ~500 bp +F ~200 bp +R 10. Analyze PCR reactions by gel electrophoresis & pb 2. pc & pd & pkanb 4. pd & pkanc 5. positive control 6. negative control 7. ladder pb pc pd ~500 bp ~500 bp kanmx4 pkanb pkanc pd ~500 bp ~500 bp

10 & pb pc & pd & pkanb pd & pkanc positive control negative control ladder Genomics Research Agenda IV Enzymatic Assays III Molecular Preparations III pb pc ~500 bp pd Aseptic Cell Culture ~500 bp II kanmx4 pkanb ~500 bp pkanc Safety and Basic Operations pd ~500 bp Research Fellowships! Laboratory Issues Application Opens March 1, Closes March 22 (do it now) ~ June 1 to ~ August 1, Select Patrick Killion As Reference Half-Time Fellowships (~ $1250) 8 weeks, 20 hours per week... or... 5 weeks, 32 hours per week Full-Time Fellowships (~ $2500) 8 weeks, 40 hours per week I

11 Lab Issues & Progress RPR II & III Due: Tuesday, March 3:30PM Hand In Now or Mailbox... Hold off on RPR IV Proposal... Lab Open & Mentors Present 10am to 8pm (M, W, Th, F) Tuesdays for Quick Maneuvers... Research Resource Calendars (Bench & PCR) Ladder Issues... Must Read Posts... Notebook Evaluation #2... RPR III Cell Pellet Process Change: Before: Final suspension in 50 ul. Now: Final suspension in 20 ul. Before: 40 ml, split to 2 x 15 ml, split to 4 x 1 ml Now: 40 ml, split to 2 x 15 ml, split to 2 x 1 ml EtOH Issues: Proper wash, spin-down process... Weekend Hours... RPR II & III Due Date... I due The Semester RPR I RPR II RPR III RPR IV Laboratory Log II&III due spring break spring break spring break through rest of semester...