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What are the aims of P6?

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What are the aims of P5?

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What are the aims of P4?

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1) The final goal of P3 is to determine which of our white colonies from our overnight incubation contained the LDHA insert. On the gel, when a band in (any of) our plasmid wells (wells 4 to 7) corresponds to the band from the well with double-digested plasmid (well 3), it means the LDHA insert is present.

2) Firstly, we isolated plasmids from the white colonies, we prepared plasmids from 4 different colonies in case some of the white colonies did not contain our LDHA insert. Next, the plasmids underwent RE digest such that we hope to get fragments containing LDHA gene separated from the main plasmid. Lastly, we run the samples on gel electrophoresis to determine which of the plasmid samples had contained the LDHA insert.

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Main aims: In general, we wanted to ligate the digested vector (with LDHA) into the RT-PCR DNA, after which we allowed the competent cells to undergo transformation to take up the vector with the LDHA insert.

Experiments: Clean-up of RE digested fragment was first done so that we had only the DNA (from RT-PCR). Next, we ligated to plasmid vector into the DNA, with the aid of ligation buffer and T4 DNA ligase. Lastly, competent DH5a cells were added to DNA, heat shocked and left to be incubated. After which, we spread the competent cells (who hopefully have transformed successfully) onto agar plates containing X-gal and IPTG, to observe for blue and white colonies in the coming week.

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Main aims: Confirm amplification of LDHA gene from RT-PCR via gel electrophoresis. Purification of RT-PCR product, followed by RE digest of RT-PCR product (LDHA) and vector.

Experiments: Gel Electrophoresis. DNA purification of RT-PCR product (LDHA) --> RE digest of RT-PCR products (LDHA) and vector

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Main aim: To extract RNA from cells which is needed in Reverse Transcription to obtain cDNA. cDNA is then used as a template to obtain multiple copies via PCR.

Experiments: RNA isolation --> Reverse Transcription --> PCR

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QUESTIONS What to key in? 1) Main aims of current practical 2) Experiments that were done 

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QUESTIONS What to key in? 1) Main aims of current practical 2) Experiments that were done 

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To obtain purifed LDHA protein using extracted total RNA from mouse fibroblast cells