1. Analysis of Gene Expression of Arabidopsis using RT-PCR and DNA Cloning Presented by Neha Jain ABE Workshop 2006 June 30, 2006

2. AIM: To isolate and sequence the cDNA of PDI2 and GFP - Extraction of total RNA from Arabidopsis - RNA template cDNA Amplify using PCR - Run gel using RT-PCR products - Purification and quantification of the cDNA - Ligation of PCR product into a TA vector - Amplification of vector DNA in bacteria Introduction RT

3. Methods Isolation of total RNA (rRNA, tRNA, mRNA) - Pulverize the leaves into a fine powder - Lysis of the cell under denaturing conditions - Centrifuged to separate the RNA from other cellular materials - The lysate is applied to a column that binds RNA - The RNA is eluted with water

4. Reverse Transcription Reverse Transcriptase (RT) – a DNA polymerase enzyme that uses an RNA template to synthesize a complementary molecule of double stranded DNA We used Moloney murine leukemia virus (M-MLV) for the synthesis of the first cDNA strand mRNA Oligo dT primer cDNA

5. Methods Reverse Transcription - The following samples were used: The concentration of RNA added to tubes 1-4 was 2μg Group12345 1,4WTPDI2 A- 1 PDI2 A- 2 No RTControl- No RNA 2,3WTpBIN35 S-GFP pGFP 2SC-1 No RTControl- No RNA

6. Methods The following reagents were added to the RNA samples (which contain template mRNA) 1. RT Buffer 2. RNase-free DNase 3. dNTPs 4. Oligo dT Primers 5. RNase-free water 37ºC – 30 min Activate DNase 70ºC – 5 min Inactivate DNase

7. RT Reaction and PCR RT: PCR: 42ºC - 1 hour Add RT 95ºC - 5 min Template DNA PCR buffer MgCl 2 dNTPs Gene specific primers DNA Polymerase PCR Product Run on gel 95ºC 5 min denature 95ºC – 30 sec 56ºC – 30 sec 72ºC – 1 min 72ºC 5 min Anneal and elongate 35 CYCLES Anneal and elongate

8. Results of RT-PCR Group 1 Group 4 100bp + WT 2A1 2A2 –RT H2O P+

9. Summary of RT-PCR Results Groups 1 and 4 LaneSampleBand? G1 G4 Explanation or Expected Result 2Henry’s RT-PCR Positive Control Henry’s RT-PCR 3WTX WT has the PDI2 gene 4PDI2 A-1X Homozygous – PDI2 gene knocked out 5PDI2 A-2X Heterozygous – should see band 6(-) No RTX No RT 7(-) Control H 2 0X No RNA template 8Henry’s Plasmid Control PDI2 inserted into plasmid

10. Results of RT-PCR 100bp + WT 2SC GFP5er –RT H2O P+ Group 2 100bp + WT 2SC GFP5er –RT H2O P+ Group 3

11. Summary of RT-PCR Results Groups 2 and 3 LaneSampleBand? G2 G3 Explanation or Expected Result 2Henry’s Positive Control Henry’s RT-PCR 3WTX GFP absent in WT 4GFP2SCX Band expected - transgenic plant 5GFP5er (35S) GFP inserted into plant 6(-) No RTX No RT 7(-) Control H 2 0X No RNA template 8Henry’s Plasmid Control GFP inserted into plasmid

12. Methods Extraction and Purification of DNA from the Agarose Gel - Cut the selected DNA fragment from the agarose gel - Melted the gel - The sample was applied to a column where the DNA binds to the membrane of the column - The DNA is eluted with buffer - The DNA can then be cloned using plasmid vectors

13. RT-PCR Products Selected for TOPO Cloning MW Hen Pl GFP H+ pBIN GFP WT Pl+ PDI2 PDI2 35S 35S pla Gr 1 Gr2 Gr3 Gr4 Gel purified RT- PCR products for use as inserts in cloning

14. Molecular Cloning What is Molecular Cloning? Refers to making multiple copies of a gene or fragment of DNA by insertion of the DNA fragment into a vector which can then replicate in a host cell Requirements for Cloning Foreign DNA - gel purified RT-PCR products Vector DNA - pCR4-TOPO plasmid vector Host Organism - E. coli

15. Overview of DNA Cloning Using a Plasmid Vector

16. TOPO TA Cloning Uses DNA Topoisomerase I - it cleaves and re-joins DNA during replication - functions as a restriction enzyme and a ligase - covalently bonded to 3' deoxythymidine (T) that overhangs a linearized plasmid - complements the 3' deoxyadenosine (A) that overhangs the PCR product

17. Methods - TOPO TA Cloning Ligation of the product into a TA Plasmid Vector - add the gel purified PCR product into 2 tubes and H 2 0 in the third as a control - add the pCR4 TOPO Vector to each tube - add salt solution to each tube which helps to ligate the DNA H 2 0 PCR Product 1PCR Product 2 pCR4 TOPO Vector

18. Plasmid Vector

19. Transformation of E. coli Transformation – the uptake of foreign DNA by bacterial cells The TOPO vector with the insert is added to “competent” E. coli The E. coli are made “competent” by treating them with ice-cold CaCl 2 followed by heat-shock at 42ºC S.O.C medium is added to the bacteria and then incubated at 37ºC for 1 hour during which transformation takes place

20. Amplification of the Recombinant Plasmid in Bacteria Host bacteria are spread on a selective nutrient medium and incubated at 37ºC overnight The plates contain ampicillin which allow the bacteria containing the recombinant plasmid to proliferate forming colonies E. coli divide every 22 minutes forming a billion cells in less than 11 hours The recombinant plasmids also replicate as the bacteria divide

21. Selection of Cells Containing Insert Insertional Mutagenesis - The LacZ gene in the plasmid codes for β-galactosidase - Xgal is a substrate for the enzyme which turns from colorless to blue White Colonies - β-galactosidase not produced due to insert Blue Colonies - β-galactosidase produced as there is no insert

22. Selection of Cells Containing Insert pCR4-TOPO Vector - the lethal E. coli ccdB gene in the pCR4-TOPO plasmid is responsible for killing the cells without an insert

23. Ligation Efficiency of RT-PCR Products into the pCR4 Vector Number of Colonies Volume plated Group1Group 2Group 3Group 4 Water Negative 10 uL 52609677 50 uL 220171258225 Ligation 1 10 uL 83 (Henry positive) 136 (Henry positive) 65 (Henry positive) 45 (wild type) 50 uL 672 (Henry positive) 600 (Henry positive) 440 (Henry positive) 145 (wild type) Ligation 2 10 uL 98 (pdi2) 107 (35Ser) 33 (35Ser) 17 (white) 6 (blue) 50 uL 412 (pdi2) 515 (35Ser) 207 (35Ser) 513 (pdi2)

24. Ligation Efficiency of RT-PCR Products into the pCR4 Vector Each group picked 10 white colonies from both ligation 1 and 2 and placed them into 10 different tubes containing LB and Ampicillin which were cultured overnight Results GroupNumber of tubes with growth Total number of tubes 1910 21 31 44 Total1540

25. Any Questions?

26. References Becker, W. M., Kleinsmith L.J., Hardin, J. (2006). The world of the cell, San Francisco: Pearson Education http://www.bio.davidson.edu/courses/Molbio/MolStudents/spring99/bill/invitrogen.ht ml http://www.blc.arizona.edu/INTERACTIVE/recombinant3.dna/clones.html http://en.wikipedia.org/wiki/Cloning_vector http://www.vivo.colostate.edu/hbooks/genetics/biotech/enzymes/rt.html http://www.invitrogen.com/content/sfs/manuals/topota_man.pdf