Long Weekend


Hello all you lovely VDSers. I hope you all had a weekend.

Last week I FINALLY got my PCR to work!!! Holy cannoli, am I behind. So, this week I worked on my scaled-up PCR and I started the actual protocol for pNIC-Bsa4 Cloning. On Friday, I followed the “Protocol of PCR for pNIC-Bsa4 Cloning”, but I multiplied all the volumes by two. I chose my ‘Sample C’ from my first PCR to scale-up. That Sample C consisted of14.25ul of the ‘Master Mix’ which had (17.5ul 10x- KOD Buffer #1, 26.25ul dNTP, 28ul of both FOR and REV primers), 2ul of MgCl2, 6.5ul of H2O, and 1.25 ul of DNA Template).  So, I basically just doubled all of those amounts and made Samples A-F of Sample C.

The first one of that came out like this: (Scaled-up PCR of Sample C)

Yes I know. It’s really bright. I realized later that I keep forgetting to dilute the KOD DNA Polymerase. If you look at Lane 10, it should be empty, but it isn’t. That’s because some of Sample F (of Sample C) spilled over. I didn’t think too much about that until Saturday when I came in and asked Leighana what she thought. She told me that I was supposed to save 1/2 of my PCR samples for PCR Cleanup 😦 I always forget to do things. Speaking of which, I forgot to not add the DNA template to Sample F. I know, I’m ridiculous.

I also worked ion preparing pNIC-Bsa4 as an accepting vector, as previously mentioned. I added used pNIC-Bsa4 plasmid that had an initial concentration of 228ng/ul. I only needed 2.25ng of that, so that came out to needing only 9.86ul of the pNIC-Bsa4 plasmid along with2.5ul of Buffer3, 0.25ul of BSA, 1ul of BSAI, and 11.39ul of Nanopure H20. I left that at 50C for 3 hours, then did PCR cleanup on it hping to work on it on Saturday.

So, on Saturday, I did another PCR with diluted KOD and I ONLY used 25ul out of the 50ul samples. Everything went well except for the fact that I received some news from home and decided to head back to my apartment. Thanks Leighana for being there. This is a picture of my PCR from Saturday (Finally done right!!!)

So that was that.

Today, I was so READY to keep working on my cloning. I was going to begin the ‘Cohesive End Generation’ steps for both my PCR and my Accepting Vector. I got everything ready and then realized that I have no Sucrose+KAN plates. Even if Ihad wanted to do it yesterday, there was no mentor to do it with. So, hopefully Dr. Beckham can help me with that.

As of now, I have my preparation done for pNIC-Bsa4 as an Accepting Vector, and I am ready to move on as soon as I have a mentor. For now, I have a presentation to work on for tomorrow! I hope I can keep you guys awake!

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2 Responses

  1. Gabriela, good job. I would recommend actually only running about 10 ul of each of your PCR samples and saving all of the rest for PCR cleanup. Keep me posted on your progress on cloning.

    • Thanks Dr. Beckham. I sent you an e-mail a while ago about my cloning. Just get back to me when you can! Thanks!

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