sample="quota" bates="2023141738" isource="pm" decade="1990" class="ui" date="19920301" VED-1 March 1, 1992 ACTION PLAN 1992 1. IMMUNOSCREENING OF EXPRESSION LIBRARY 2. Use aminoacid sequence information to synthesize primers for use in PCR. 3. USE PCR to isolate desired sequences from the tobacco root RNA and DNA. a. RNA PCR (Perkin-Elmer kit,Forward and Reverse primers) b. DNA PCR (Perkin-Elmer kit,Forward and Reverse Primers) c. ANCHORED PCR: AMPLIFICATION WITH SINGLE-SIDED SPECIFICITY 1. Take tobacco root poly A RNA.Synthesise complementary strand of DNA using oligo dT as primer .Destroy RNA by alkali hydrolysis.Amplify Using combinations of forward primers and oligo dT25.CLONE the PCR PRODUCT.SEQuence the insert. Enter the sequence into the computer.use DNA STAR(findpro programme) to find PMT1.PRO,PMT2.PRO, PMT3.PRO PMT4.PRO sequences. 2.ANCHORED PCR OF cDNA with OLIGO(dG) tails.Synthesise cDNA using REVERSE PCR PRIMERS or oligo dT. Add oligo dG to the newly synthesized c DNA strand.Amplify using REVERSE PCR PRIMERS and OLIGO dC.Clone the PCR product AND analyse as in 1. d.LIGATION-MEDIATED ANCHORED PCR.Digest the genomic DNA with a Mbo1 OR its isoschizomer Sau3A. If possible, Fill one base to prevent self-Ligation. LIgate an anchor-primer on the ends of the DNA fragments. Amplify with a specific primer as well as the anchor. The choice of which strand the primer is derived from determines which side of the fragment is amplified. This can be done for several enzymes that leave cohesive ends with a four-base 5'overhang. This could allow isolation of the promoter for the gene of interest. 4. GENERATION OF SINGLE-STRANDED LABELED PRIMERS AND THEIR USE IN NORTHERN AND SOUTHERN HYBRIDIZATION AND SCREENING OF LIBRARIES. 5. PMT POLYCLONALS (GPC FRACTION) IMMUNOSCREENING OF cDNA EXPRESSION LIBRARY. 1. EXPRESSION LIBRARY OF TOBACCO ROOT mRNA IS AT HAND. 2. A POLYCLONAL ANTIBODY AGAINST A SYNTHETIC PEPTIDE IN N-29 MER HAS BEEN RAISED 3. THIS POLYCLONAL ANTIBODY LIGHTS UP 60 KD REGION IN WESTERN BLOTS OF TOBACCO PROTEIN. 4. THE EXPRESSION LIBRARY (1) WILL BE SCREENED USING POLYCLONAL ANTIBODY (2) 5. IF A POSITIVE CLONE IS FOUND, THE PROTEIN OF THAT CLONE MAY BE PURIFIED USING ANTI betaGAL ANTIBODIES. PCR AND PRIMERS by VED MALIK 1.Importance of first PCR cycle AND 3' end of primers 2.Important to know where you are 3.Examination of PMT. SEquences. 4. N-termainal codons included in primers 5.C-terminal codons should appear in amplified product. 6.C-terminal codons can constitute the primers,and N-terminal codons can appear in the PCR product. formulaname formulaname formulaname Fig 6. Comparison of the location of regions of sequence similarity of the human D-Asp/L-isoAsp and other methyl-transferases along their polypeptide chains. The homology regions, connected by vertical lines, are: Region I (diagonal shading), Region II (dotted shading), and Region III (diamond shading). Sequences are detailed in Tables IX (Region I) and X (Regions II and III). Although not listed, Region I is located at a similar position in the other bacterial RNA adenine methyltransferases. ACTION PLAN 1992 THESE ARE ONLY SUGGESTED PLANS. PRIORITY DECREASES WITH THE INCREASING ASSIGNED NUMBER 1. Obtain aminoacid sequence ( c-terminal) of PMT. 2.Use aminoacid sequence information to synthesize primers for use in PCR. 3.USE PCR to isolate desired sequences from the tobacco root RNA and DNA. a. RNA PCR(Perkin-Elmer kit,Forward and Reverse primers) b. DNA pcr(Perkin-Elmer kit,Forward and Reverse Primers) c. ANCHORED PCR:AMPLIFICATION WITH SINGLE-SIDED SPECIFICITY 1.Take tobacco root poly A RNA.Synthesize complementary strand of DNA using oligo dT as primer .Destroy RNA by alkali hydrolysis.Amplify Using combinations of forward primers and oligo dT25 .CLONE the PCR PRODUCT.SEQuence the insert. Enter the sequence into computer use.DNA STAR (findpro programme) to find PMT1.PRO, PMT2.PRO, PMT3.PRO PMT4.PRO sequences. 2.ANCHORED PCR of cDNA with OLIGO(dG)tails.Synthesise cDNA using REVERSE PCR PRIMERS or oligo dT. Add oligo dG to the newly synthesised c DNA strand.Amplify using REVERSE PCR PRIMERS and OLIGO dC.Clone the PCR product AND analyse as in 1. d. LIGATION-MEDIATED ANCHORED PCR. Digest the genomic DNA with a Mbo1 OR its isoschizomer Sau3A. If possible, Fill one base to prevent self-Ligation. LIgate an anchor-primer on the ends of the DNA fragments. Amplify with a specific primer as well as the anchor. The choice of which strand the primer is derived from determines which side of the fragment is amplified. This can be done for several enzymes that leave cohesive endswith a four-base 5'overhang. This could allow isolation of the promoter for the gene of interest. 4. GENERATION F SINGLE-STRANDED LABELED PRIMERS AND THEIR USE IN NORTHERN AND SOUTHERN HYBRIDIZATION 5.IMMUNOSCREENING OF EXPRESSION LIBRARY> GENERATION OF TRANSGENIC PLANTS CURRENT STATUS After 10-14 days culture, separate developing calli and transfer to fresh medium As soon as morphologically acceptable shoots appear, transfer shoots to rooting medium containing 500 mg/L carbenicillin and 100 mg/L kanamycin After rooting, transgenic plantlets can be transferred to greenhouse or maintained as meristem cultures indefinitely until greenhouse space is available LEAF DISC TRANSFORMATION Grow transformed bacterial clones overnight in suspension culture Excise leaf material from axenic Burley 21 plantlets and cut lamina into 5-10 mm sections Dip sections into cell culture, blot on sterile gauze and transfer to shoot induction medium without antibiotics After 2 days incubation (28ºC, 16 hr photoperiod, 5000 lux), rinse sections in 500 mg/L carbenicillin, blot and transfer to shooting medium with 500 mg/L carbenicillin and 300 mg/L kanamycin Electroporate cells at 100 µF capacitance, 250 µsec discharge interval, 480 v, .3mm gap for final field strength of 16000 v/cm Dilute cells 10-fold with nonselective culture medium and incubate at rt for 30-60 minutes Plate 10µl and 70 µl on Luria + 100 µg/ml kanamycin After incubating for 2-3 days at 28ºC, select 3 isolates from each construct and generate pure cultures for leaf transformation AGROBACTERIUM ELECTROPORATION Grow overnight suspension culture of Agrobacterium tumefaciens str. LBA4404 Wash cells 2x in 1mM Hepes + 10% glycerol with a final wash in 10% glycerol alone (reduce volume to 500 µl) Mix 2 µl of DNA with 25 µl cells Transfer 8 µl of mix to Hoefer bacterial electrode (5.6mm) GENERATION OF TRANSGENIC PLANTS VIA PLANT TISSUE CULTURE GENERATION OF TRANSGENIC PLANTS VIA PLANT TISSUE CULTURE Ask and Ye shall Receive Ask and Ye shall Receive GENERATION OF TRANSGENIC PLANTS VIA PLANT TISSUE CULTURE