Lab Report/Assignment.
1) Annotate the gel photo of your own gel and provide a succinct figure legend
Ans:
The results of the nested PCR have been shown in Figure 1a.
The procedure adopted was as follows. The germline, splenocyte and thymocyte DNA samples were taken to perform a nested PCR with two sets of primers Set 1 contains external primers and nested primers for DHQ52 and JH4, while Set2 contains external and nested primers for DHFL16.1 and JH4. The first round of PCR was performed with an external couple of Set1 and Set2 primers and subsequently, the second round of PCR was performed by nested primers of Set1 and Set2, after taking 1/10th of the volume of the PCR product from the first round as a template. For each PCR a 100ng of each primer was taken in a total reaction volume of 20Î¼l. The DNA amount taken in the first PCR was 100ng and in the second PCR, the 1Î¼l product from the first reaction was used. PCR conditions used were, 94Â°C for 2 minutes followed by 15 cycles of denaturation (94Â°C 30seconds), annealing (63Â°C, 30 seconds) and extension (68Â°C, 2 minutes) followed by the last step of 68Â°C for 5 minutes. PCR product mixed with 6X loading dye and was then loaded on 1.8% agarose gel for electrophoresis and stained with Ethidium bromide to visualize the bands. The loading pattern was as follows:
Lane1: First PCR reaction from DHQ52 primers and germline DNA
Lane2: First PCR reaction from DHQ52 primers and Splenocyte DNA
Lane3: First PCR reaction from DHQ52 primers and Thymocyte DNA
Lane4: First PCR reaction from DHFL16.1 primers and germline DNA
Lane:5 First PCR reaction from DHFL16.1 primers and Splenocyte DNA
Lane6: First PCR reaction from DHFL16.1 primers and Thymocyte DNA
Lane7: 1Kb DNA marker
Lane8: Second PCR reaction from DHQ52 primers and germline DNA
Lane9: Second PCR reaction from DHQ52 primers and Splenocyte DNA

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