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Lab meeting 13.02.25

Lab meeting 13.02.25. Summary (Lab meeting 27.01. 2013). No mutation was detected in K562, Hela, IM9. Step1 : Primers for cloning of splicing gene.

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Lab meeting 13.02.25

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  1. Lab meeting 13.02.25

  2. Summary (Lab meeting 27.01. 2013) No mutation was detected in K562, Hela, IM9

  3. Step1 : Primers for cloning of splicing gene • Kozak sequence is recognized by the ribosome as the translational start site, from which a protein is coded by that mRNA molecule. The ribosome requires this sequence to initiate translation. • C-myc used to separate recombinant by using antibody of C-myc and detection by Western blotting.

  4. Step 1: Amplification products using Ex-Tag • PCR products: As most PCR products amplified with TaKaRa ex Taq™ have one A added at the 3'-termini, the obtained PCR product can be directly used for cloning into a T-vector. • U2AF1: 510 bp U2AF1 K562 IM9 HeLa 1000 bp 500bp

  5. Step 2: pGEM-T Easy vector circle map and Sequence reference points

  6. Step 2: Cloning U2AF1 of K562, IM9 and Hela into pGEM-T vector

  7. Step 2: Cloning U2AF1 of K562, IM9 and Hela into pGEM-T vector K562 has 12 white colonies Hela has 9 white colonies IM9 has 15 white colonies

  8. Step 3: Examine successful colonies and prepare for cloning into expression vector • LA broth culture • Plasmid extraction • Direct sequencing

  9. Lab meeting 13.03.04 • K562: 2 successful colonies : No. 1&7 (2/12) • Hela: 1 successful colonies : No. 8 (1/9) • IM9: 2 successful colonies : No. 5&8 (2/15) DNA sequencing confirmed that the reconstructed plasmid pGEM-T easy vector contained the sequence cDNA of the U2AF1 gene, which was composed of exons 1 to 9, 177 aa and 552 bp. 

  10. cDNA U2AF1 digested with NheI, XhoI cDNA of U2AF1 gene was composed of exons 1 to 9, 177 aa and 552 bp.  Source: http://tools.neb.com/NEBcutter2/showdig.php?name=e051c50b-U2AF1-cloning_seq_C-

  11. Restriction Endonuclease NheI, XhoI After digested by RE NheI & XhoI • Restriction Digestion for Analysis Reaction • NheI 0.3 µl • XhoI 0.3 µl • 10X NEBuffer 1 µl • BSA 0.5 µl • pGEM-T + cDNA U2AF1 (40-46ng/ µl) 1 µl • Distilled water 6.9 µl • Total: 10 µl • Incubation time: 1 hour • Incubation temp: 37 °C K562 HelaIM9 No.1 No.7 No.8 No.5 No.8 3000 bp pGEM-T easy vector (3012 bp) 500 bp Digested cDNA U2AF1 (546 bp) Ready for ligation into pcDNA3.1

  12. Plasmid pcDNA3.1 extraction Uncut pcDNA 3.1 (5428bp) 1kb plus DNA ladder 5000 bp Ready for digested with RE NheI, XhoI

  13. pcDNA3.1 digested with NheI, XhoI Source: http://tools.neb.com/NEBcutter2/showdig.php?name=a4b78c98-pcDNA_3.1_seq

  14. Restriction Endonuclease NheI, XhoI Digested pcDNA 3.1 (5338bp) Uncut pcDNA 3.1 (5428bp) 1kb plus DNA ladder • Restriction Digestion for Analysis Reaction • NheI 0.3 µl • XhoI 0.3 µl • 10X NEBuffer 1 µl • BSA 0.5 µl • pcDNA3.1 (55ng/ µl) 1 µl • Distilled water 6.9 µl • Total: 10 µl • Incubation time: 1 hour • Incubation temp: 37 °C 5000 bp Ready for ligation with cDNA U2AF1

  15. Step 4: Clonne into expression vector pcDNA3.1 • Gel purification of cDNA U2AF1 gene • pcDNA3.1-cDNA U2AF1 Ligation

  16. Lab meeting 13.03.11 Condition for ligation reaction • Restriction Digestion for Ligation Reaction • NheI 2 µl • XhoI 2 µl • 10X NEBuffer (1X) 5 µl • BSA 3 µl • pGEM-T + cDNA U2AF1 (40-46ng/ µl) 2 µl • Distilled water 36 µl • Total: 50 µl • Incubation time: overnight • Incubation temp: 37 °C • Restriction Digestion for Ligation Reaction • NheI 2 µl • XhoI 2 µl • 10X NEBuffer (1X) 5 µl • BSA 3 µl • pcDNA3.1 (55ng/ µl) 2 µl • Distilled water 36 µl • Total: 50 µl • Incubation time: overnight • Incubation temp: 37 °C

  17. Ligation Reaction • 2X Rapid Ligation buffer, T4 DNA ligase 5 µl • pcDNA3.1 vector (55ng/ µl) 2 µl • cDNA U2AF1 (2 ng/ µl) 10 µl • T4 DNA ligase (3 units/µl) 2 µl • Distilled water 1 µl • Total: 20 µl • Incubation time: overnight • Incubation temp: 4°C

  18. Ligate the insert into the appropriate vector and transform into E. coli. • Selecttransformants on LB plates containing 100 μg/ml ampicillin. • Analyze your transformants for the presence of insert by restriction digestion. • Select a transformant with the correct restriction pattern and use sequencing to confirm that your gene is cloned in the proper orientation.

  19. Resequencing Human Mitochondrial DNA with the mitoSEQr • mtDNA: • maternally inherited circular 16569 bp • Encoding 13 subunits of the respiratory chain • 2 rRNAs • 22 tRNAs • No introns, but contains a non-coding control region • The control region regulates the replication and transcription of the mtDNA and is known for its highly variable regions.

  20. Resequencing Human Mitochondrial DNA with the mitoSEQr • Applied Biosys, Innovations, July 08 • Two resequencing sets • MitoALL™-46 resequencing amplicons for the whole mtDNA. >98% coverage. • MitoCR™–9 resequencing amplicons for the control region. 100% coverage. • 5 amplicons shared between the two sets • 500 (10 μL) reactions

  21. Resequencing Human Mitochondrial DNA with the mitoSEQr • mitoSEQr system • PCR-based resequencing system • Identification of sequence variations • Entire mito genome • Control region • Methodology • Overlapping regions amplified with specific primer pairs • Tailed with universal M13 sequences and designed for universal PCR and Sequencing conditions. • Generates re-sequencing amplicons

  22. Resequencing Human Mitochondrial DNA with the mitoSEQr • MitoSEQr™ Procedure • Genomic DNA • PCR reaction • PCR Reaction Clean-up • Sequencing Reaction • Sequencing Reaction Clean-up • Electrophoresis • Data Analysis

  23. Resequencing Human Mitochondrial DNA with the mitoSEQr • Identifying mitochondrial mutations • Heteroplasmy-mixture of more than one type of an organellar genome • Symptoms of severe heteroplasmic mito disorders frequently do not occur until adulthood • Role of mitochondrial in diseases, including cancer and Alzheimer’s

  24. Resequencing Human Mitochondrial DNA with the mitoSEQr • Oncocytoma: characterized by proliferation of mitochondria • Sequence analysis identified specific mutations causing oncocytic tumors • Homoplasmic frameshift mutation in ND1, in a cell model of thyroid oncocytic tumor • ND1, one of the 45 complex I subunits of the respiratory chain • SAME mutation, and other similar mutations in complex I, detected in vivo in biopsies from oncocytic tumors of different origins: thydroid, breast and kidney

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