"
Friday, August 26
From 2011.igem.org
(Purification of 9 samples in LB Broth shaken since Thurs around 10pm) |
|||
| Line 1: | Line 1: | ||
| - | Dr Liz working alone in lab on Friday | + | ==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! | 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. | 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! | ||
| + | |||
| + | 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. | ||
Revision as of 07:17, 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!
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.
