Pdi11 (3) pdi1 (2) pdi2 (2) pdi4 (2) pdi6 (2) pdi7 (2) pdi9 (1) pdi3 (2) pdi5 (3) pdi8 (0) pdi10 (1) GeneAccession #'s Chr #T-DNA knockoutComments Pdi1At3g54960.

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Presentation transcript:

Pdi11 (3) pdi1 (2) pdi2 (2) pdi4 (2) pdi6 (2) pdi7 (2) pdi9 (1) pdi3 (2) pdi5 (3) pdi8 (0) pdi10 (1) GeneAccession #'s Chr #T-DNA knockoutComments Pdi1At3g54960 CAB41088 AL Chr3Salk_ A Salk_ B Pdi2At5g60640 BAB09837 AB Chr5Salk_ A Salk_ B Subjects Pdi3At1g52260 AAG51554 AC Chr1Salk_ A Sail_302_G10 B Pdi4At3g16110 BAB02677 AB Chr3Salk_ A Salk_ B Pdi5At1g21750 AAD41430 AC Chr1Salk_ Salk_ Salk_ Pdi6At1g77510 Q9SRG3 AC Chr1Salk_ A Salk_ B Pdi7At4g27080 T06038 T24A18.30 Chr4Salk_ A Salk_ B Pdi8At1g35620 AAK62431 AF Chr1 Pdi9At2g32920 AAB91984 AC Chr2Salk_ A Sail_595_D09 not availabl Pdi10At1g04980 AAF40463 AC Chr1Sail_548_F05 Pdi11At2g47470 O22263 AY Chr2Salk_ A Salk_ B Salk_ C

Genotype - Segregation T-DNA in the Arabidopsis Genome T-DNA – (transfer DNA) the transforming region of DNA in the Ti plasmid of Agrobacterium tumefaciens. It is the vehicle to deliver foreign genes to plant cells. T-DNA T-DNA Flanking Region LBRB KAN Pdi2 1:3

PCR Amplified Fragments Cloning and Sequencing How to Map Your T-DNA ? GSP T-DNA Flanking Region GSP LB RB T-DNA

Results and Discussion What is a wild type PCR band? What PCR band patterns indicate that a plant is a heterozygote or homozygote? M - + N pr2pr1 pr1 pr3 Normal Mutant Hm Ht Hm

PCR Mapping of T-DNAs in Pdi2 Gene R1 primer Wild Type Primer F1 Pdi2 A LB T-DNA 800bp WT ~ 620 bp T-DNA Predicted PCR products F1/LB F1/R1

Materials and Solutions: DNA samples and PCR solutions (?) Procedures: 1. Set up PCR (see Protocol I). 2. Check PCR products using agarose gels (see Protocol II). T-DNA Mapping Experiment

PCR Reaction Mix Reaction mix: 1 X6 X DNA template (50-100ng) 1.0 ul --- ddH2O18.1 ul x Immolase buffer, 2.5 ul15.0 dNTP (25 mM), 0.2 ul 1.2 MgCl2(25mM) 2.0 ul12.0 Primer 1 (100ng/ul) 0.5 ul 3.0 Primer 2 (100ng/ul) 0.5 ul 3.0 Immolase DNA Polymerase 0.2 ul 1.2 (5 U/ul) Total volume 25 ul24 X 6 DNA samples: WT, PDI2A-1, PDI2A-2, Positive (+) and Negative control (H2O)

PCR Conditions PCR conditions: 95  C for 7 min 35 cycles 95  C for 30 seconds Tm – 5  C, 30 seconds 72  C, extension 1 min/kb 72  C, 7 min 4  C, ~~ Immolase is inactive at room temperature; it needs heat activation at 95  C. Annealing temp : Tm- 5  C

T-DNA Mapping Pdi2 A: SALK_ F1/R2 (WT Band) WTA1 A2 WT 800bp F1/LB (T-DNA Band) WTA1 A 2WT Annealing Temp 55  C MW