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Friday, August 26
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First, everything was kept very cold. I even put a vortexer in the coldroom next to the microfuge. Hint: If you know you're going to be working in the cold room for any extended period of time, make sure you are wearing warm socks and shoes. I'd even recommend a wool hat. By the time I was done with these cold steps in the purification process, I felt like a human popsicle! | First, everything was kept very cold. I even put a vortexer in the coldroom next to the microfuge. Hint: If you know you're going to be working in the cold room for any extended period of time, make sure you are wearing warm socks and shoes. I'd even recommend a wool hat. By the time I was done with these cold steps in the purification process, I felt like a human popsicle! | ||
| + | I followed the protocol mentioned above, taking 1.5 ml aliquot from each cell culture sample, and putting the remainder back into the cold room. I used Solution I and II, already available in the fridge and freezer from earlier in the month when I had some alone time in the lab, and mixed a fresh batch of Solution II, as recommended. I did not have 10 M NaOH -- only 0.4 M NaOH stock solution was available. I also had 10% SDS solution -- a different batch than that used to purify the P, R and T parts. See the Section called "Growing up a cell culture ..." below in this page. that which Steve, Patrick and I used the Thurday night that we stayed late, purifying the P, R and T BioBricks that had been made using the parts from our Spring Distribution Kit, and competent cells (Top 10 -One Shot), followed by plating on amp plates and grown up using a single colony per tube containing LB broth + amp. . I made roughly enough for the nine smaples. | ||
Second: The phenol:chloroform step of the purification process is labelled as optional. However, the authors of Handbook, as well as other molecular biologists who have posted information on the web about the phenol:chloroform extraction step seem to agree with this: Downstream reactions may not work as well if you do not do the phenol:chloroform step. | Second: The phenol:chloroform step of the purification process is labelled as optional. However, the authors of Handbook, as well as other molecular biologists who have posted information on the web about the phenol:chloroform extraction step seem to agree with this: Downstream reactions may not work as well if you do not do the phenol:chloroform step. | ||
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| + | ==Growing up a cell culture seeded by a single colony from a Cloned cell== | ||
| + | After the P, R and T parts were eluted by Patrick and other team members, they were used to clone competent cells, which were then plated on agarose +amp plates. Single colonies were chosen from the plates after incubation, and were grown up in amp-rich LB broth in the warm shaker. One thing we learned since then is that parafilm is not to be used when shaking a cell culture overnight - the idea is to get air in there. We were concerned about the culture sloshing out of the tube, because the lid was loose, and understood only that air is required for cell growth, but thought sufficient air would be in the tube if we filled each tube with cell culture taking up no more than 1/3 of the tube's volume. That might explain some things in cases where there was no DNA on gels run after purification of the plasmid DNA from those cell cultures. We DID NOT make this error on with the 9 cell cultures put in the incubator on Thursday night, August 25. | ||
Revision as of 07:45, 30 August 2011
Dr Liz working alone in lab on Friday
CAUTION - AS THE WIKI NOTES THAT I TYPE KEEP GETTING DELETED BY SOME INADVERTENT ACTION ON MY PART (WARNING - DON'T USE THE BACK BUTTON), I WILL BE SAVING THIS PAGE THROUGHOUT THE EDITING PROCESS, BECAUSE i CAN'T STAND THE IDEA OF $#@%! TYPING EVERYTHING FOR A fourth TIME TONIGHT!
The cell cultures in LB broth + amp that had been shaking in the incubator overnight were removed to the cold room about 5:00 pm. This amounted to about 17 hours, and we had set the incubator low - 31.5 degrees C. So I expect they were still in log growth phase.
Purification Protocol
I used the Phage DNA purfication by cell lysis protocol from the Handbook of Molecular Cloning, Vol 1. Jessica has input this protocol on the wiki- I have not found it yet. Please link this page to the protocol if you can, somebody.
There were a couple of modifications and special points worth discussing about using this protocol, as opposed to using spin tubes in a kit.
First, everything was kept very cold. I even put a vortexer in the coldroom next to the microfuge. Hint: If you know you're going to be working in the cold room for any extended period of time, make sure you are wearing warm socks and shoes. I'd even recommend a wool hat. By the time I was done with these cold steps in the purification process, I felt like a human popsicle!
I followed the protocol mentioned above, taking 1.5 ml aliquot from each cell culture sample, and putting the remainder back into the cold room. I used Solution I and II, already available in the fridge and freezer from earlier in the month when I had some alone time in the lab, and mixed a fresh batch of Solution II, as recommended. I did not have 10 M NaOH -- only 0.4 M NaOH stock solution was available. I also had 10% SDS solution -- a different batch than that used to purify the P, R and T parts. See the Section called "Growing up a cell culture ..." below in this page. that which Steve, Patrick and I used the Thurday night that we stayed late, purifying the P, R and T BioBricks that had been made using the parts from our Spring Distribution Kit, and competent cells (Top 10 -One Shot), followed by plating on amp plates and grown up using a single colony per tube containing LB broth + amp. . I made roughly enough for the nine smaples. Second: The phenol:chloroform step of the purification process is labelled as optional. However, the authors of Handbook, as well as other molecular biologists who have posted information on the web about the phenol:chloroform extraction step seem to agree with this: Downstream reactions may not work as well if you do not do the phenol:chloroform step.
Growing up a cell culture seeded by a single colony from a Cloned cell
After the P, R and T parts were eluted by Patrick and other team members, they were used to clone competent cells, which were then plated on agarose +amp plates. Single colonies were chosen from the plates after incubation, and were grown up in amp-rich LB broth in the warm shaker. One thing we learned since then is that parafilm is not to be used when shaking a cell culture overnight - the idea is to get air in there. We were concerned about the culture sloshing out of the tube, because the lid was loose, and understood only that air is required for cell growth, but thought sufficient air would be in the tube if we filled each tube with cell culture taking up no more than 1/3 of the tube's volume. That might explain some things in cases where there was no DNA on gels run after purification of the plasmid DNA from those cell cultures. We DID NOT make this error on with the 9 cell cultures put in the incubator on Thursday night, August 25.
