Friday, August 26

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Revision as of 08:17, 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 that includes Phenol:Chloroform Step

I used the Phage DNA purfication by 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 are a couple of modifications and special points worth discussing about using this protocol, as opposed to using spin tubes in a kit.

First: Everything must be and 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 on the following point: Downstream reactions may not work as well if you do not do the phenol:chloroform step. In particular, restriction digestion steps may not work well. One solution to a slow restriction digestion is to simply overload the amount of the restriction enzyme in the reaction - as much as a 10-fold increase is used in cases requiring better results. According to NEB, this is a fairly standard procedure, when needed. As we live on donations, it seemed to make sense to just do the phenol chloroform step instead. More information, particularly safety information, about doing this step of the purification is found below in the "Phenol:Chloroform" section.

The Basic Purification By Lysis Protocol, using Solutions I, II and III

I began the purification by following the protocol mentioned above, taking a 1.5 ml aliquot from each cell culture sample (each originally contained ~3.0 ml), 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. and mixed a fresh batch of Solution II, as recommended. I used 0.4 M NaOH stock solution as it was what was available (as opposed to 10 M NaOH, which is recommended). The 10% SDS solution was also freshly made by Cheryl Brown within the last month -- a different batch than that used to make the Solution II that was used in this basic protocol to purify the P, R and T parts. I made roughly enough for the nine samples.

An interesting observation was that there appeared to be significantly more of cellular material in the bottoms of all of the 3B tubes compared to the tubes with 4A as part of their label. This note was made after the addition of the 3 solutions, the vortexing and the accompanying spindown that pelleted that material.

Keeping everything ice cold, all steps were done up to the step where 90% ethanol would be added to precipitate the DNA from the supernatant obtained in the previous step. The phenol:chloroform steps were done before this ethanol precipitation step. After the phenol:chloroform step, the DNA precipitation was accomplished using 90% ethanol, follwed by a spin (always at 12,000 rpm at 4 degrees C through all steps except the phenol steps), pouring off of the 90% ethanol while being mindfull of the DNA pellet, followed by a final wash with 70% ethanol and the accompanying final spindown and pouring off of this last ethanol wash, carefully avoiding accidently pouring away the often-loose pellet of DNA at the bottm of the tube. See the "Finding Your Pellet after Centrifugation" section below.

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.

@@ Line 9: / Line 9: @@
 I used the Phage DNA purfication by 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.
+There are 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!
+'''First''': Everything must be and 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 on the following point: Downstream reactions may not work as well if you do not do the phenol:chloroform step. In particular, restriction digestion steps may not work well. One solution to a slow restriction digestion is to simply overload the amount of the restriction enzyme in the reaction - as much as a 10-fold increase is used in cases requiring better results. According to NEB, this is a fairly standard procedure, when needed. As we live on donations, it seemed to make sense to just do the phenol chloroform step instead. More information, particularly safety information, about doing this step of the purification is found below in the "Phenol:Chloroform" section.
+===The Basic Purification By Lysis Protocol, using Solutions I, II and III===
+I began the purification by following the protocol mentioned above, taking a 1.5 ml aliquot from each cell culture sample (each originally contained ~3.0 ml), 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. and mixed a fresh batch of Solution II, as recommended. I used 0.4 M NaOH stock solution as it was what was available (as opposed to 10 M NaOH, which is recommended). The 10% SDS solution was also freshly made by Cheryl Brown within the last month -- a different batch than that used to make the Solution II that was used in this basic protocol to purify the P, R and T parts. I made roughly enough for the nine samples.
-I began the purification by following 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. and mixed a fresh batch of Solution II, as recommended. Using 0.4 M NaOH stock solution as it was what was available. The 10% SDS solution was also freshly made by Cheryl Brown within the last month -- a different batch than that used to purify the P, R and T parts. I made roughly enough for the nine smaples.
+An interesting observation was that there appeared to be significantly more of cellular material in the bottoms of all of the 3B tubes compared to the tubes with 4A as part of their label. This note was made after the addition of the 3 solutions, the vortexing and the accompanying spindown that pelleted that material.
 Keeping everything ice cold, all steps were done up to the step where 90% ethanol would be added to precipitate the DNA from the supernatant obtained in the previous step.  The phenol:chloroform steps were done before this ethanol precipitation step. After the phenol:chloroform step, the DNA precipitation was accomplished using 90% ethanol, follwed by a spin (always at 12,000 rpm at 4 degrees C through all steps except the phenol steps), pouring off of the 90% ethanol while being mindfull of the DNA pellet, followed by a final wash with 70% ethanol and the accompanying final spindown and pouring off of this last ethanol wash, carefully avoiding accidently pouring away the often-loose pellet of DNA at the bottm of the tube. See the "Finding Your Pellet after Centrifugation" section below.