I think I'm doing my project with a snail speed!
In the past month, since the last week in September, speak in a more exact way, I kept doing PCR day by day. I was glad to do it since I needed not to make more solutions, but I didn't mean I wanted to do the EXACTLY same thing everyday!
Gosh, my PCR didn't work for rather a long period!
Everything seemed alright. I didn't think I can even make mistakes on weighting stuff and put it into water according to the desired concentration. I adjusted my solutions to the proper pH. I did positive controls of my PCR using others' buffers. But, no matter what I did, my OWN buffer didn't work! What the hell!
I started to doubt everything, even the water I used.
Anyways,
Calm down, water wouldn't be a problem!
I read the recipe again. Hold on! It says “pH 8.4 for Taq buffer, if use pH 7.5 ones, both of Taq and PWO would work......”
Okay, that's the point.
I made some Tris-HCl in pH of 8.4. Then, PCR again...
Gosh, it worked perfectly...
The damn pH-issue cost me almost one month to solve it. Fine, I won't trust all protocols too much from now on... -__-
At least, I confirmed that I was not that bad on making solutions. XDD
In the past month, since the last week in September, speak in a more exact way, I kept doing PCR day by day. I was glad to do it since I needed not to make more solutions, but I didn't mean I wanted to do the EXACTLY same thing everyday!
Gosh, my PCR didn't work for rather a long period!
Everything seemed alright. I didn't think I can even make mistakes on weighting stuff and put it into water according to the desired concentration. I adjusted my solutions to the proper pH. I did positive controls of my PCR using others' buffers. But, no matter what I did, my OWN buffer didn't work! What the hell!
I started to doubt everything, even the water I used.
Anyways,
Calm down, water wouldn't be a problem!
I read the recipe again. Hold on! It says “pH 8.4 for Taq buffer, if use pH 7.5 ones, both of Taq and PWO would work......”
Okay, that's the point.
I made some Tris-HCl in pH of 8.4. Then, PCR again...
Gosh, it worked perfectly...
The damn pH-issue cost me almost one month to solve it. Fine, I won't trust all protocols too much from now on... -__-
At least, I confirmed that I was not that bad on making solutions. XDD
Finally, time to try some new experiments. Transcription! Yay~
However, I was a bit nervous on it. I remembered the last time I extracting RNA. It was degraded totally. Fortunately, I only needed a short fragment, thus the degradation didn't matter. However, now it was all about RNA, would my RNA be safely extracted and kept?
I started my transcription. I added my PCR product and all other stuff needed. When I did PCR, all stuff was just DNA-grade. I knew it, yet I still used my PCR product directly in the transcription. Afterwards, I extracted the RNA. I loaded the final purified RNA onto a regular agarose gel. I put the gel into UV...
I saw bands!
My RNAs were all safe! Good!
At that time, I was a bit amazed by the “cleanness” of my lab. I used DNA-grade stuff in RNA-level experiments, and I got what I wanted! That's so wonderful!
Or, would I say it's only because RNAse hasn't found its way to me?
However, I was a bit nervous on it. I remembered the last time I extracting RNA. It was degraded totally. Fortunately, I only needed a short fragment, thus the degradation didn't matter. However, now it was all about RNA, would my RNA be safely extracted and kept?
I started my transcription. I added my PCR product and all other stuff needed. When I did PCR, all stuff was just DNA-grade. I knew it, yet I still used my PCR product directly in the transcription. Afterwards, I extracted the RNA. I loaded the final purified RNA onto a regular agarose gel. I put the gel into UV...
I saw bands!
My RNAs were all safe! Good!
At that time, I was a bit amazed by the “cleanness” of my lab. I used DNA-grade stuff in RNA-level experiments, and I got what I wanted! That's so wonderful!
Or, would I say it's only because RNAse hasn't found its way to me?