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| See [[User:JCAnderson|this page]] for info about me. | | See [[User:JCAnderson|this page]] for info about me. |
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| ===061606===
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| pAC997 came out just fine. We today mapped several constructs moved from their original pSB1A* plasmids into pAC997. Those constructs all are named pSB3C6-*. Monday we are splitting the group into two subgroups, one doing conjugation (Samantha/Jennifer) and the other doing riboregulators (Bryan/Kaitlin). For riboregulators, we still need to make p15A variants of both traJF and traJR(const. promoters).
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| The traJF construct (J01093) was fine on paper but the subclones just didn't go apparently. We're redoing that one by PCR. We are still trying to rescue a construct (J01024 from -80, but the first one just didn't grow) of traJR. I think it might be a plasmid backbone questionmark issue there. We also did TlamOlam (F knockout strain) competent cells today, still will need to do Tlam (R cells) competent cells. I realized today that I have no idea what plasmid the oriTF plasmids J01002 are in. So, we're also spotting the two -80 stocks with that part name to see what they are. there is also a 1064 part in the -80, and we spotted that too to see if it is usable. We may have to do some subcloning there. We'll see tomorrow. Also need to check on the status of the oriTR plasmid. Hmmm. So, that's the state of conjugation.
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| For the riboregulators, transferring lock3-RFP (J01122) into pSB3C6 went smoothly. We also transferred the constitutive, unlocked variant (OnRFP, J01022) into the p15A. The colonies were pink on plates, so that's a good sign that we'll be able to do libraries and positive screens on plates. So, we went ahead and did the EIPCR for a library of key3 variants. Doing the 944 library described in construction files to see how it goes. We blunted out the XbaI site of the pSB1A2-key3 plasmid with Vent (product pSB1A2-J01129X) and setup EIPCR today.
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| ===060706===
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| I ran a gel on the pAC977 EIPCR:
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| [[Image:060706a.jpg|thumb|pAC977 EIPCR]]
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| Looks fine, digesting BamHI/DpnI in NEB2, will ligate and transform MC1061 today.
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| ===060606===
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| The team arrived yesterday, and we began assessing the status of our stocks and determining the parts we'd need to do the first experiments. Today we did a lot of colony growing, replating, restreaking, and miniprepping. We set up one experiment to determine the full antibiotic resistance biotope for TlambdaOlambda and Rlambda, the F and R plasmid knockout strains. We need this to figure out what markers we can use on our plasmids. In addition, we received oligo ca997 (and some other ones described in my personal construction file 060206, they'll be put into the wiki as we use them). 997 is for constructing pAC977 by EIPCR. I setup the PCR this evening using the Expand kit/8K55 program. The scheme is described in the 060206 construction file.
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| Meanwhile, Bryan has setup this much more professional-looking wiki on OpenWetWare due to some difficulties we were having on the iGEM wiki page.
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| ===052606===
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| I'm trying to figure out this wiki thing. This is where science notes should be.
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