Day 13
Day 13 - Let the project begin
When Zhen Dong came to the lab in the morning, he asked me where were the embryos. I told them I only had 10 (which were the remaining embryos that I had not given to Shen Qian and Foong Mei) and he sort of scolded me. The previous day he told me to collect at least 20. Then, I told him that I had isolate some RNA from lots of embryo yesterday. So I just pray that my RNA had a decent quality.
I waited for Shen Qian and Foong Mei before I ran my gel to see the RNA bands. And once again, I made a mistake. I loaded DNA ladder instead of RNA ladder but fortunately, the result was good. At least I got 2 clear band and 1 fuzzy band. the 2 clear band corresponded to rRNA while the fuzzy band at the lower part of the gel correspond to the tRNA. The smear at the band was the mRNA.
After that, we proceed with reverse transcription-PCR (RT-PCR) to isolate and amplify the desired mRNA. In RT-PCR, complementary DNA (cDNA) was produced using RNA as a template, therefore the name Reverse Transcription. After RT-PCR, we performed PCR using the primer that Zhen Dong and I have designed (the sample) and Beta-actin primer as our positive control. We left our PCR in the machine overnight as we were short of time.
When Zhen Dong came to the lab in the morning, he asked me where were the embryos. I told them I only had 10 (which were the remaining embryos that I had not given to Shen Qian and Foong Mei) and he sort of scolded me. The previous day he told me to collect at least 20. Then, I told him that I had isolate some RNA from lots of embryo yesterday. So I just pray that my RNA had a decent quality.
I waited for Shen Qian and Foong Mei before I ran my gel to see the RNA bands. And once again, I made a mistake. I loaded DNA ladder instead of RNA ladder but fortunately, the result was good. At least I got 2 clear band and 1 fuzzy band. the 2 clear band corresponded to rRNA while the fuzzy band at the lower part of the gel correspond to the tRNA. The smear at the band was the mRNA.
After that, we proceed with reverse transcription-PCR (RT-PCR) to isolate and amplify the desired mRNA. In RT-PCR, complementary DNA (cDNA) was produced using RNA as a template, therefore the name Reverse Transcription. After RT-PCR, we performed PCR using the primer that Zhen Dong and I have designed (the sample) and Beta-actin primer as our positive control. We left our PCR in the machine overnight as we were short of time.
Labels: RNA gel electrophoresis, RNA isolation, RT-PCR
posted by Melinda Leonie @ 12:05 AM
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