lab 5 DGD - BCH 3356 English Thursday 8:30-10:00 TBT333...

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BCH 3356 English – Thursday 8:30-10:00 TBT333 English – Friday 2:30-4:00 MNT203 Megan Rose –
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Lab 1 PCR amplification of fragment of interest (T7 RNA polymerase gene) from parent plasmid. T7 RNA polymerase insert PCR amplification Forward primer Reverse primer
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Lab 2
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Lab 3 Transform E. coli cells with ligation products. Plate transformation mixtures on agar with ampicillin (pTrcHisB possesses a gene for antibiotic resistance). Only successfully transformed cells with survive and grow.
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Lab 4 Inoculated and screened five transformant colonies to confirm the presence of the T7 insert into the pTrcHisB/T7 plasmid. Sequenced the T7 insert from your positive clone to identify the point mutation that had been introduced in the coding sequence
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Timelime for the second half Lab 5 (November 7-10): Protein expression: Inoculation, induction and cell harvest Lab 6 (November 14-17): Protein purification: His-tag protein purification by affinity chromatography
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Lab 5 Reinoculation of the positive transformant cell line (selected during lab 4) into a liquid culture and IPTG- induction for overexpression of T7 RNA polymerase. AMP IPTG
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Lab 6 After induction, the cells will be harvested (centrifuged) and lysed in order to extract the T7 RNA polymerase protein. Resuspend cells in lysis buffer. Pellet cells Pellet cell debris Protein will be in supernatan t
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Lab 6 All of the bacterial proteins are in the supernatant, so the T7 RNA polymerase will be further purified by His-tag affinity chromatography His-tag Affinity chromatography SDS-PAGE Supernatant before after
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Perform a Western Blot analysis using an antibody specifically raised against the His-tag. Only the fusion proteins with the his-tags will
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lab 5 DGD - BCH 3356 English Thursday 8:30-10:00 TBT333...

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