Team:MIT/Notebook

From 2014.igem.org

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<h2>miRNA</h2>
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<div  style="padding:20px">
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6.22<br>
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<table width="90%" align="center">
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Update the parts list on the wiki<br>
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Create an experiments page on the wiki<br>
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Prepare to meet with Jake Beal<br>
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Re-transform MAV1212<br>
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Re-transform MAV1212-Hef1a-eBFP2<br>
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Mini prep MAV1212-Hef1a-eBFP2<br>
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6.23<br>
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Go over workflow of plasmid construction as a group<br>
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Finish a unified daily log<br>
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Digest meeting with Jake Beal<br>
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Go through plates<br>
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Re plate MAV1212<br>
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Pick colonies for MAV1212<br>
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Plan LacZ PCR product<br>
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Primers ( ? )<br>
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Figure out what's going on<br>
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<br>
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6.24<br>
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Present about cell models<br>
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COMPLETE PARTS LIST ON WIKI<br>
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Prep experiments<br>
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Contact Jeremy about multiple miRNA target sites chained together<br>
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Pick colonies for MAV1212<br>
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Miniprep MAV1212<br>
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Keep MAV1212-Hef1a-eBFP2 (for use WITHOUT target sites)<br>
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Gateway MAV1212-Hef1a-L7ae (for high sensor)<br>
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Gateway MAV1212_Hef1a-eYFP (for use WITH target sites)<br>
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Transform Hef1a, MAV1212 ( ? )<br>
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NOTES:<br>
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No MAV1212 colonies grew on the plates or in the medium<br>
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Jeremy gave us more, enough for one more transformation<br>
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6.25<br>
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Create a clear workflow<br>
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Update "status" of plasmids on parts list<br>
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How do we string multiple low sensors together?<br>
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Determine which pairs of target sites we want<br>
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Find sequences (hopefully under 200 bp)<br>
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Send ultramer order to Brian<br>
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Tranform LRs <br>
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MAV1212-Hef1a-mKate2<br>
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MAV1212-Hef1a-L7ae<br>
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Hef1a on Kan<br>
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Pick colonies from transformations<br>
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MAV1212-Hef1a-eBFP2<br>
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Golden Gate<br>
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Gather miRNA target sites from Jeremy<br>
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Make all 6 single low sensors<br>
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NOTES<br>
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MAV1212 didn't grow (ccdB is over expressed)<br>
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Hef1a was plated on Amp plates...but it has Kan resistance<br>
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Verify MAV1212-Hef1a-eBFP2 via sequencing AmpR/eBFP2 region<br>
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<br>
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6.26<br>
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AD blood cells from PrecisionMed? Talk to Brian?<br>
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Meet with Kyle about designing siRNA (email sent)<br>
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Make siRNA sequences in geneious<br>
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Send order to Brian<br>
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Picture of how low sensors come together<br>
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Determine which pairs of target sites we want<br>
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Find sequences (hopefully under 200 bp)<br>
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Send ultramer order to Brian<br>
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Plan new MAV vector<br>
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Plan high sensor construction (meet with Brian)<br>
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-
Golden Gate<br>
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Gather miRNA target sites from Jeremy<br>
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Nanodrop for concentrations ( ? )<br>
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Make all 6 single low sensors (in conjunction with antibody group)<br>
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Miniprep eBFP2<br>
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NOTES<br>
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Both LR's failed (mKate2 and L7ae)<br>
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Meeting w/ Jeremy tomorrow to talk about new MAV vector and high sensors<br>
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We need to Gibson in  eBFP2 before we can finish the sensors<br>
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<br>
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siRNA MEETING WITH KYLE<br>
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mature (5' phospho) and sense (reverse complement)<br>
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copy format from 2013<br>
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send brian both sequences as a duplex (IDT has a duplex option), with all the modification<br>
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We NEED to Gibson in the eBFP2 control<br>
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each high sensor on its own transcript (not necessarily own plasmid)<br>
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QUESTIONS<br>
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what do the "r" and "m" stand for?<br>
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-rna and modification<br>
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where do the overhangs come from? why are they there?<br>
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-mature one has 2 u's (constant)<br>
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6.27<br>
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Write up low sensor experiment plans<br>
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Keep updating the parts list<br>
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Meet with Jeremy<br>
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Discuss high sensor plans<br>
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New MAV assembly plan<br>
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Questions for Brian<br>
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eBFP2 Gibson for Flow Cytometry ( ? )<br>
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Send siRNA sequences<br>
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Miniprep Hef1a<br>
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Golden Gate all the low sensors<br>
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NOTES<br>
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Reasons to not use phages for circuit delivery<br>
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6.30<br>
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Keep updating the parts list<br>
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Strengthen experiments page<br>
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Prepare protocol for building new MAV<br>
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Prepare protocol for building high sensors<br>
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Transform low sensor Golden Gates<br>
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Start building new MAV<br>
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<br>
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<br>
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NOTES<br>
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Meet w/ Brian tomorrow to talk about double input ultramer<br>
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Co-transfect 2x Kturn & MAV w/ target sites & eBFP2<br>
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PCR for building new MAV <br>
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<br>
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7.1<br>
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Keep updating the parts list<br>
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Meet w/ Brian about double input low sensor<br>
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Fill out PCR protocol to be ready for tomorrow<br>
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Create high sensor construction map (unified to look like the other groups)<br>
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Design primers / find restriction enzymes to sequence low sensors<br>
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Pick multiple low sensor colonies<br>
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Re-transform low sensors<br>
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NOTES<br>
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Double input low sensor will be a gBlock<br>
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Lots of blue colonies, not many white colonies (none from GH)<br>
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<br>
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7.2<br>
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Keep updating the parts tree<br>
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Propose experiments to test L7ae and Tau14/21<br>
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Find restriction enzymes to digest original EF<br>
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Miniprep original EF<br>
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PCR mRFP out of pL1F_1_S6_S7<br>
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PCR Purify mRFP<br>
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Run the gel for mRFP<br>
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Re Golden Gate EF and GH<br>
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Digest mRFP cassette<br>
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Re ligate mRFP cassette<br>
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Transform mRFP cassette<br>
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Retransform EF and GH<br>
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NOTES<br>
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There were no colonies to pick from EF and GH --> re transform<br>
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<br>
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7.7<br>
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Keep updating the parts tree (include experiments)<br>
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Update experiments page<br>
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Obtain plasmid maps<br>
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Email Jin about TAL14 and miRNA target sites<br>
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Talk to Lyla/Alexa about transfecting later this week<br>
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Look for plasmids<br>
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pEXPR: UAS-Gal4-TAL14-eYFP<br>
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pEXPR: Gal4VP16<br>
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pEXPR: Hef1a-TAL14<br>
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pEXPR: Hef1a-2x Kturn-eYFP<br>
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Transformations<br>
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Low Sensor Golden Gates<br>
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pEXPR: Hef1a-eBFP2 (186 ng/uL)<br>
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pEXPR: Hef1a-L7ae (96 ng/uL)<br>
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Pick red colonies and grow in medium!<br>
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Find concentrations of eBFP2 and L7ae<br>
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NOTES<br>
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Nelson doesn't know where it is, but he needs it as well, so he's on the hunt<br>
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If we're ready, we can transfect very early next week<br>
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<br>
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7.8<br>
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Keep updating the parts tree (include experiments)<br>
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Figure out where the Golden Gates went<br>
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Create protocol for siRNA transfection<br>
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Meet with Jin at 3<br>
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Transformations <br>
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-
EF<br>
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GH<br>
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Mutant Hef1a-2xKturn-eGFP<br>
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Wild Type Hef1a-2xKturn-eGFP<br>
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Pick colonies from L7ae and eBFP2<br>
+
-
Miniprep MAV1212<br>
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Digest MAV1212<br>
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Run gel for MAV1212<br>
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Image gel for MAV1212<br>
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NOTES<br>
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Jin gave us ideas to optimize our high sensors and experiments<br>
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<br>
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7.9<br>
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Keep updating the parts tree (include experiments)<br>
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Find new miRNA target site vector. Email:<br>
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Kyle<br>
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Lila<br>
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Samira<br>
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Obtain plasmid maps<br>
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Hef1a-2xKturn (mutant and w.t.)<br>
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GAL4 (from Brian)<br>
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Pick colonies & Grow in medium<br>
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EF<br>
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GH<br>
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pENTR: L7ae<br>
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pEXPR: Hef1a-eBFP2<br>
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Mutant Hef1a-2xKturn-eGFP<br>
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Wild Type Hef1a-2xKturn-eGFP<br>
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MAV12112-Hef1a-eBFP2 (control)<br>
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Create pENTR: TAL14<br>
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BP Reaction from TAL14<br>
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Transform TAL14<br>
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NOTES<br>
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GH and pENTR: L7ae didn't grow properly (their plates had almost no agar)<br>
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L7ae was re transformed<br>
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<br>
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7.10<br>
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Keep updating the parts tree (include experiments)<br>
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More descriptive names<br>
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Include other plasmid<br>
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Alternative backbones? Check iGEM 2013<br>
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Plan experiment to test Kturn (Just Kturn and L7ae)<br>
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Re-make double input low sensor as two ultramers (use the Q3 site)<br>
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Pick colonies & grow in medium (pick 5)<br>
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Midi prep<br>
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EF<br>
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GH<br>
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pEXPR: Hef1a-eBFP2<br>
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-
Mutant Hef1a-2xKturn-eGFP<br>
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Wild Type Hef1a-2xKturn-eGFP<br>
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Mini prep<br>
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pENTR: L7ae<br>
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GH<br>
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Mini prep<br>
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EF<br>
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pEXPR: Hef1a-eBFP2<br>
+
-
Mutant Hef1a-2xKturn-eGFP<br>
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Wild Type Hef1a-2xKturn-eGFP<br>
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LR<br>
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MAV1212-Hef1a-L7ae<br>
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MAV1212-Hef1a-TAL14<br>
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BP<br>
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P4-donor-P1R on Brian's bench<br>
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TAL14-mKate pEST in Brian's fridge<br>
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Golden Gate<br>
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GH<br>
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Transformation<br>
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TAL14 Promoter (from BP rxn)<br>
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TAL14-mKate-pest<br>
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pENTR: L1_TAL14_L2<br>
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MAV1212-mRFP<br>
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NOTES<br>
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We should sequence the low sensors when they're done<br>
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Golden Gate Calculations<br>
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<br>
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7.11<br>
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Keep updating the parts tree (include experiments)<br>
+
-
Alternative backbones? Check iGEM 2013<br>
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Actual list of experiments<br>
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TAL14 & GAL4 (to test promoter)<br>
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MAV1212-hEF1a-eBFP2<br>
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Make/Find primers<br>
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To sequence both low sensors<br>
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Sequence/digest plasmids to verify - may be unnecessary. Emailed Brian.<br>
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EF<br>
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GH - don't have this<br>
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pEXPR: MAV1212-Hef11a-eBFP2<br>
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Mutant Hef1a-2xKturn-eGFP<br>
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Wild Type Hef1a-2xKturn-eGFP<br>
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Pick colonies and grow up<br>
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MAV1212-mRFP<br>
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L1_TALER14_L2<br>
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pTAL14-mKate-pEST<br>
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MAV1212-Hef1a-eBFP2 for midiprep (pending Brian's approval)<br>
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EF low sensor for midiprep (pending Brian's approval)<br>
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Mutant Hef1a-2xKturn-eGFP for midiprep (pending Brian's approval)<br>
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WT Hef1a-2xKturn-eGFP for midiprep (pending Brian's approval)<br>
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Miniprep<br>
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GH - don't have this<br>
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Transformations<br>
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GH (Golden Gate)<br>
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Hef1a-L7ae (LR)<br>
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Hef1a-TAL14 (LR)<br>
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BP<br>
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P4-donor-P1R with TAL14-mKate pEST<br>
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NOTES<br>
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BP rxn didn't yield colonies --> re-doing it and letting it grow overnight this time<br>
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GH plate has no colonies :/<br>
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<br>
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7.14<br>
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Keep updating the parts tree (include experiments)<br>
+
-
Obtain pEXPR: hEF1a-GAL4VP16<br>
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Fill out the Genewiz Order Form to sequence Low Sensor miR-144<br>
+
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Prepare transfection well plate maps<br>
+
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So where are the siRNA though...<br>
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Why didn't the GH golden gate work on try #3?<br>
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Change name of high sensors in tree<br>
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Pick colonies<br>
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pEXPR: TAL14-mKate<br>
+
-
Midiprep<br>
+
-
pEXPR: MAV1212-Hef11a-eBFP2<br>
+
-
Mutant Hef1a-2xKturn-eGFP<br>
+
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Wild Type Hef1a-2xKturn-eGFP<br>
+
-
Low Sensor miR-144<br>
+
-
Hef1a-L7ae (LR)<br>
+
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Miniprep<br>
+
-
MAV1212-mRFP<br>
+
-
L1_TALER14_L2<br>
+
-
pTAL14-mKate-pEST<br>
+
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LR<br>
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pEXPR: MAV1212-TRE-L7ae<br>
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NOTES<br>
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so...the siRNA are nowhere to be found<br>
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hEF1a-TAL14 (LR) has no colonies<br>
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TRE is nowhere to be found<br>
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<br>
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7.15<br>
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Keep updating the parts tree (include experiments)<br>
+
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Obtain Plasmids<br>
+
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pEXPR: hEF1a-GAL4VP16<br>
+
-
pENTR: TRE (promoter)<br>
+
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pEXPR: TAL14-mKate<br>
+
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Troubleshoot<br>
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So where are the siRNA though... (keep looking around, they were ordered)<br>
+
-
Why didn't the GH golden gate work on try #3?<br>
+
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Retransform products at higher concentration<br>
+
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Fill out the Genewiz Order Form to sequence Low Sensor miR-144<br>
+
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Design primers to sequence high sensors<br>
+
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Ask Jeremy about sequencing primers<br>
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Look into siRNA transfection<br>
+
-
TOP PRIORITY: SEQUENCE LOW SENSORS (can't be done till primers arrive)<br>
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Pick colonies<br>
+
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pEXPR: TAL14-mKate<br>
+
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LR<br>
+
-
pEXPR: MAV1212-TRE-L7ae<br>
+
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Transfection<br>
+
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L7ae experiment<br>
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Golden Gate<br>
+
-
High Sensor miR-30d (I13 & J13)<br>
+
-
High Sensor miR-146a (I5 & J5)<br>
+
-
High Sensor let-7f (1.13 & 1.14)<br>
+
-
High Sensor miR-125b (A3 & B3)<br>
+
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Combined Low Sensor miR-144-miR-181c<br>
+
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Get ultramers from Brian<br>
+
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Anneal target sites<br>
+
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All high sensors<br>
+
-
Transformation<br>
+
-
Re-do Low Sensor miR-181c<br>
+
-
Midiprep<br>
+
-
pEXPR: TAL14-mKate<br>
+
-
NOTES<br>
+
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The transfections are a little bit messed up<br>
+
-
We need to verify the MAV1212-hEF1a-L7ae<br>
+
-
<br>
+
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7.16<br>
+
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How To Please Ron/Brain<br>
+
-
Keep updating the parts tree (include experiments)<br>
+
-
miRNA Sensors (Theory)<br>
+
-
Anaylze Low Sensor miR-144 sequencing results<br>
+
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miRNA Sensors (Lab Work)<br>
+
-
Restriction Digest<br>
+
-
MAV1212-hEF1a-L7ae / that other shit in the fridge<br>
+
-
Golden Gate<br>
+
-
High Sensor miR-30d (I13 & J13)<br>
+
-
High Sensor miR-146a (I5 & J5)<br>
+
-
High Sensor let-7f (1.13 & 1.14)<br>
+
-
High Sensor miR-125b (A3 & B3)<br>
+
-
Low Sensor miR-144 (E5 & F5)<br>
+
-
Low Sensor miR-181c (G7 & H7)<br>
+
-
Low Sensor miR-144-miR-181c<br>
+
-
Transform<br>
+
-
pEXPR: MAV1212-TRE-L7ae (LR)<br>
+
-
pEXPR: MAV1212-hEF1a-GAL4VP16 (LR)<br>
+
-
pEXPR: MAV1212-TRE-GAL4VP16 (LR)<br>
+
-
Re-suspend Low Sensor miR-144-181c oligos<br>
+
-
Anneal Low Sensor miR-44-181c oligos<br>
+
-
NOTES<br>
+
-
Low Sensor miR-144 did not pass quality control for sequencing<br>
+
-
After annealing the double input low sensors, the didn't nanodrop well<br>
+
-
Golden Gate Calculations<br>
+
-
Restriction Digest Calculations<br>
+
-
<br>
+
-
7.17<br>
+
-
�<br>
+
-
Keep updating the parts tree (include experiments)<br>
+
-
Troubleshoot<br>
+
-
Why didn't the oligos nanodrop??<br>
+
-
miRNA Sensors (Lab Work)<br>
+
-
Golden Gate<br>
+
-
Low Sensor miR-144-miR-181c<br>
+
-
Transform<br>
+
-
High Sensor miR-30d<br>
+
-
High Sensor miR-146a<br>
+
-
High Sensor let-7f<br>
+
-
High Sensor miR-125b<br>
+
-
Low Sensor miR-144<br>
+
-
Low Sensor miR-181c<br>
+
-
Pick colonies to miniprep and midiprep<br>
+
-
pEXPR: MAV1212-TRE-L7ae (LR)<br>
+
-
pEXPR: MAV1212-hEF1a-GAL4VP16 (LR)<br>
+
-
pEXPR: MAV1212-TRE-GAL4VP16 (LR)<br>
+
-
Prepare and run a gel (Hyperladder 2 and 4% agaros gel w/ annealed target sites)<br>
+
-
High Sensor miR-30d TS<br>
+
-
High Sensor miR-146a TS<br>
+
-
High Sensor let-7f TS<br>
+
-
High Sensor miR-125b TS<br>
+
-
Low Sensor miR-144 TS<br>
+
-
Low Sensor miR-181c TS<br>
+
-
FACS cell prep<br>
+
-
Transfect L7ae Experiment<br>
+
-
Analyze FACS data<br>
+
-
NOTES<br>
+
-
We have a new primer to sequence with (again, from Jeremy)<br>
+
-
We now have FACS DATA<br>
+
-
7.18<br>
+
-
Keep updating the parts tree (include experiments)<br>
+
-
Troubleshoot<br>
+
-
Why didn't the oligos nanodrop??<br>
+
-
Meet w/ Kyle about FlowJo<br>
+
-
Look for the siRNA<br>
+
-
Golden Gate<br>
+
-
Low Sensor miR-144-miR-181c<br>
+
-
Pick colonies<br>
+
-
High Sensor miR-30d<br>
+
-
High Sensor miR-146a<br>
+
-
High Sensor let-7f<br>
+
-
High Sensor miR-125b<br>
+
-
Low Sensor miR-144<br>
+
-
Low Sensor miR-181c<br>
+
-
Miniprep<br>
+
-
pEXPR: MAV1212-TRE-L7ae (LR)<br>
+
-
pEXPR: MAV1212-hEF1a-GAL4VP16 (LR)<br>
+
-
pEXPR: MAV1212-TRE-GAL4VP16 (LR)<br>
+
-
Restriction Digest & run gel<br>
+
-
pEXPR: MAV1212-TRE-L7ae (LR)<br>
+
-
pEXPR: MAV1212-hEF1a-GAL4VP16 (LR)<br>
+
-
pEXPR: MAV1212-TRE-GAL4VP16 (LR)<br>
+
-
Midiprep?<br>
+
-
pEXPR: MAV1212-TRE-L7ae (LR)<br>
+
-
pEXPR: MAV1212-hEF1a-GAL4VP16 (LR)<br>
+
-
pEXPR: MAV1212-TRE-GAL4VP16 (LR)<br>
+
-
Gateway<br>
+
-
pEXPR: MAV1212-hEF1a-TAL14<br>
+
-
Transform<br>
+
-
pENTR: TAL14<br>
+
-
pENTR: TAL21<br>
+
-
pENTR: Something for yeast for Brian<br>
+
-
NOTES<br>
+
-
The gel didn't work very well, so we're re-doing it tomorrow<br>
+
-
7.20<br>
+
-
Keep updating the parts tree (include experiments)<br>
+
-
Meet w/ Kyle about FlowJo<br>
+
-
Troubleshoot<br>
+
-
What's going on with the double input low sensor<br>
+
-
Move siRNA into our cryobox<br>
+
-
Pick colonies<br>
+
-
High Sensor miR-30d<br>
+
-
High Sensor miR-146a<br>
+
-
High Sensor let-7f<br>
+
-
High Sensor miR-125b<br>
+
-
Low Sensor miR-144<br>
+
-
Low Sensor miR-181c<br>
+
-
pENTR: TAL14<br>
+
-
pENTR: TAL21<br>
+
-
pENTR: Something for yeast for Brian<br>
+
-
Restriction Digest & run gel<br>
+
-
pEXPR: MAV1212-TRE-L7ae (LR)<br>
+
-
pEXPR: MAV1212-hEF1a-GAL4VP16 (LR)<br>
+
-
pEXPR: MAV1212-TRE-GAL4VP16 (LR)<br>
+
-
Inoculate midiprep culture?<br>
+
-
If the LR's have the right sequence<br>
+
-
Transform<br>
+
-
Re transform MAV1212-hEF1a-TAL14 LR<br>
+
-
FACS Prep<br>
+
-
NOTES<br>
+
-
The digest keeps failing<br>
+
-
<br>
+
-
7.21<br>
+
-
Keep updating the parts tree (include experiments)<br>
+
-
Meet w/ Kyle<br>
+
-
FlowJo<br>
+
-
Bright Field Images<br>
+
-
miRNA Sensors (Theory)<br>
+
-
Troubleshoot<br>
+
-
What's going on with the double input low sensor<br>
+
-
Is there a different GG thermal cycler program for Bbs1?<br>
+
-
Run new transfection plate/protocol by Brian<br>
+
-
Move siRNA into our cryobox<br>
+
-
Grab sequencing primer from Jeremy's bench<br>
+
-
Pick colonies<br>
+
-
pEXPR: MAV1212-hEF1a-TAL14 (Mini)<br>
+
-
Give plates to Brian<br>
+
-
pENTR: TAL14p<br>
+
-
pENTR: TAL21p<br>
+
-
pENTR: Something for yeast for Brian<br>
+
-
Restriction Digest & run gel<br>
+
-
pEXPR: MAV1212-TRE-L7ae (LR)<br>
+
-
pEXPR: MAV1212-TRE-GAL4VP16 (LR)<br>
+
-
Transform<br>
+
-
pEXPR: MAV1212-hEF1a-GAL4VP16<br>
+
-
Golden Gate <br>
+
-
All the sensors<br>
+
-
Split and Seed 4 plates<br>
+
-
NOTES<br>
+
-
We transfected way too much DNA --> new protocol with 1 ug total<br>
+
-
<br>
+
-
7.22<br>
+
-
Keep updating the parts tree (include experiments)<br>
+
-
Meet w/ Kyle<br>
+
-
FlowJo<br>
+
-
Bright Field Images<br>
+
-
miRNA Sensors (Theory)<br>
+
-
Troubleshoot<br>
+
-
What's going on with the double input low sensor<br>
+
-
Run new transfection plate/protocol by Brian<br>
+
-
Move siRNA into our cryobox<br>
+
-
Grab sequencing primer from Jeremy's bench<br>
+
-
Miniprep<br>
+
-
pEXPR: MAV1212-hEF1a-TAL14<br>
+
-
Restriction Digest & run gel (4 samples)<br>
+
-
pEXPR: MAV1212-TRE-L7ae (LR)<br>
+
-
Transform<br>
+
-
pEXPR: MAV1212-TRE-GAL4VP16 (LR)<br>
+
-
All the low sensors<br>
+
-
Transfect for L7ae Experiment 1.3<br>
+
-
<br>
+
-
<br>
+
-
NOTES<br>
+
-
We ran out of Lipofectamine and couldn't transfect <br>
+
-
<br>
+
-
7.24<br>
+
-
How To Please Ron/Brain<br>
+
-
Keep updating the parts tree (include experiments)<br>
+
-
miRNA Sensors (Theory)<br>
+
-
miRNA Sensors (Lab Work)<br>
+
-
Midi prep<br>
+
-
pEXPR: MAV1212-hEF1a-TAL14<br>
+
-
Transform<br>
+
-
pEXPR: MAV1212-TRE-L7ae (LR from last night)<br>
+
-
Proteinase K<br>
+
-
MAV1212-hEF1a-TAL14<br>
+
-
Restriction Digest and run gel<br>
+
-
MAV1212-hEF1a-TAL14<br>
+
-
LR<br>
+
-
MAV1212-hEF1a-TAL21<br>
+
-
Mini prep<br>
+
-
Low Sensor miR-144<br>
+
-
Low Sensor miR-181c<br>
+
-
High Sensor miR-30d<br>
+
-
High Sensor miR-125b<br>
+
-
High Sensor miR-146a<br>
+
-
High Sensor let-7f<br>
+
-
Send for sequencing<br>
+
-
Low Sensor miR-144<br>
+
-
Low Sensor miR-181c<br>
+
-
High Sensor miR-30d<br>
+
-
High Sensor miR-125b<br>
+
-
High Sensor miR-146a<br>
+
-
High Sensor let-7f<br>
+
-
Pick colonies<br>
+
-
Low Sensor miR-144<br>
+
-
Low Sensor miR-181c<br>
+
-
High Sensor miR-30d<br>
+
-
High Sensor miR-125b<br>
+
-
High Sensor miR-146a<br>
+
-
High Sensor let-7f<br>
+
-
pEXPR: MAV1212-hEF1a-GAL4VP16<br>
+
-
pEXPR: MAV1212-TRE-GAL4VP16<br>
+
-
Seed plates<br>
+
-
L7ae experiment (Take 3)<br>
+
-
NOTES<br>
+
-
pEXPR: MAV1212-hEF1a-GAL4VP16 and pEXPR: MAV1212-TRE-GAL4VP16 only had blue colonies, which doesn't make sense because they were both verified...<br>
+
-
Brian is in the middle of something on his bench, so we won't midi today<br>
+
-
<br>
+
-
7.25<br>
+
-
Keep updating the parts tree (include experiments)<br>
+
-
Troubleshoot<br>
+
-
What's the deal with the GAL4VP16?!<br>
+
-
Pick colonies<br>
+
-
pEXPR: MAV1212-TRE-L7ae (mini)<br>
+
-
MAV1212-hEF1a-GAL4VP16 (midi)<br>
+
-
MAV1212-TRE-GAL4VP16 (midi)<br>
+
-
Proteinase K<br>
+
-
MAV1212-hEF1a-TAL21<br>
+
-
MAV1212-hEF1a-TAL14<br>
+
-
Transform<br>
+
-
MAV1212-hEF1a-TAL21 (LR)<br>
+
-
MAV1212-hEF1a-TAL14 (LR)<br>
+
-
Plasmids from BCR group<br>
+
-
Plasmids from protein receptor group<br>
+
-
Plasmids from Brian<br>
+
-
Miniprep<br>
+
-
All the sensors (round 2)<br>
+
-
Send for sequencing<br>
+
-
All the sensors (round 2)<br>
+
-
Analyze sequencing results<br>
+
-
All the sensors (round 1)<br>
+
-
Transfect<br>
+
-
L7ae Experiment (Take 3)<br>
+
-
Seed<br>
+
-
TAL14 Experiment (Take 1)<br>
+
-
Re-anneal<br>
+
-
miR-144-181c double input target site<br>
+
-
Golden Gate<br>
+
-
Low Sensor miR-144-181c<br>
+
-
Inoculate midi cultures<br>
+
-
Each of the single input sensors<br>
+
-
NOTES<br>
+
-
All of the single input sensors were verified!<br>
+
-
1st full day with no hiccups? <br>
+
-
Gave constructs and plasmids maps (Kturns, L7ae, and MAV RFP) to Jeremy<br>
+
-
7.26<br>
+
-
Keep updating the parts tree (include experiments)<br>
+
-
Mini prep<br>
+
-
pEXPR: MAV1212-TRE-L7ae<br>
+
-
Digest and run gel<br>
+
-
pEXPR: MAV1212-TRE-L7ae<br>
+
-
Inoculate midi (if digest is okay)<br>
+
-
pEXPR: MAV1212-TRE-L7ae<br>
+
-
Midi prep<br>
+
-
MAV1212-hEF1a-GAL4VP16<br>
+
-
MAV1212-TRE-GAL4VP16<br>
+
-
All the single input sensors<br>
+
-
Pick colonies<br>
+
-
MAV1212-hEF1a-TAL21<br>
+
-
MAV1212-hEF1a-TAL14<br>
+
-
Plasmids from BCR group<br>
+
-
Plasmids from protein receptor group<br>
+
-
Plasmids from Brian<br>
+
-
Analyze sequencing results<br>
+
-
All the sensors (round 2)<br>
+
-
Transform<br>
+
-
Low Sensor miR-144-181c<br>
+
-
Transfect<br>
+
-
TAL14 Experiment (Take 1)<br>
+
-
Seed<br>
+
-
Experiment #3 (Take 1)<br>
+
-
Experiment #4 (Take 2)<br>
+
-
NOTES<br>
+
-
<br>
+
-
7.28<br>
+
-
Keep updating the parts tree (include experiments)<br>
+
-
Include TAL14 and TAL21 high sensors<br>
+
-
Analyze sequencing results<br>
+
-
Plan DOX induction experiment for L7ae repression<br>
+
-
Plan to build TAL14/TAL21 high sensors<br>
+
-
Do gating and analysis of Exp 1 Attempt 3<br>
+
-
Mini prep<br>
+
-
pEXPR: MAV1212-TRE-L7ae<br>
+
-
Digest and run gel<br>
+
-
pEXPR: MAV1212-TRE-L7ae<br>
+
-
Inoculate midi (if digest is okay)<br>
+
-
pEXPR: MAV1212-TRE-L7ae<br>
+
-
Pick colonies<br>
+
-
MAV1212-hEF1a-TAL21<br>
+
-
MAV1212-hEF1a-TAL14<br>
+
-
Low Sensor miR-144-181c<br>
+
-
Golden Gate<br>
+
-
All the target sites & TAL14-mKate with Bbs1<br>
+
-
NOTES<br>
+
-
we're starting construction of the TAL14/21 high sensors<br>
+
-
<br>
+
-
7.29<br>
+
-
How To Please Ron/Brain<br>
+
-
Keep updating the parts tree (include experiments)<br>
+
-
Get TAL21 promoter from Brian<br>
+
-
Plan experiments<br>
+
-
DOX induction for L7ae repression (look here)<br>
+
-
Tuning TAL14 repression<br>
+
-
<br>
+
-
Learn about gating and FACS analysis<br>
+
-
Midi prep<br>
+
-
pEXPR: MAV1212-TRE-L7ae<br>
+
-
Mini prep<br>
+
-
MAV1212-hEF1a-TAL21<br>
+
-
MAV1212-hEF1a-TAL14<br>
+
-
Low Sensor miR-144-181c<br>
+
-
Sequence verify<br>
+
-
Low Sensor miR-144-181c<br>
+
-
Digest and run gel<br>
+
-
MAV1212-hEF1a-TAL21<br>
+
-
MAV1212-hEF1a-TAL14<br>
+
-
Golden Gate (if digest works)<br>
+
-
All the TAL21 high sensors<br>
+
-
Transform<br>
+
-
All the TAL14 high sensors<br>
+
-
And a plasmid from BCR<br>
+
-
NOTES<br>
+
-
The minipreps failed? re do tomorrow<br>
+
-
<br>
+
-
7.30<br>
+
-
Keep updating the parts tree (include experiments)<br>
+
-
Run tuning experiments by Brian<br>
+
-
Get transformation code from Brian (hopefully)<br>
+
-
Midi prep<br>
+
-
pEXPR: TAL21-mKate<br>
+
-
Mini prep<br>
+
-
MAV1212-hEF1a-TAL21<br>
+
-
MAV1212-hEF1a-TAL14<br>
+
-
Low Sensor miR-144-181c<br>
+
-
Sequence verify<br>
+
-
Low Sensor miR-144-181c<br>
+
-
Digest and run gel<br>
+
-
MAV1212-hEF1a-TAL21<br>
+
-
MAV1212-hEF1a-TAL14<br>
+
-
Golden Gate (if digest works)<br>
+
-
All the TAL21 high sensors<br>
+
-
Pick colonies<br>
+
-
miR-30d (TAL14)<br>
+
-
miR-125b (TAL14)<br>
+
-
let-7f (TAL14)<br>
+
-
TAL14 mKate<br>
+
-
Inoculate midi culture (in case the digest works)<br>
+
-
TAL21<br>
+
-
NOTES<br>
+
-
The midi prep for TAL21-mKate didn't work --> pick a colony and try again tomorrow<br>
+
-
High sensor miR-146a didn't grow colonies --> retransform + replate transformation<br>
+
-
<br>
+
-
7.31<br>
+
-
Keep updating the parts tree (include experiments)<br>
+
-
What about the double input low sensor? <br>
+
-
Midi prep<br>
+
-
pEXPR: TAL21-mKate<br>
+
-
Run gel<br>
+
-
MAV1212-hEF1a-TAL21<br>
+
-
MAV1212-hEF1a-TAL14<br>
+
-
Golden Gate (if digest works)<br>
+
-
All the TAL21 high sensors<br>
+
-
Gateway (LR)<br>
+
-
pEXPR: hEF1a-TAL21<br>
+
-
Pick colonies<br>
+
-
miR-30d (TAL14)<br>
+
-
miR-125b (TAL14)<br>
+
-
let-7f (TAL14)<br>
+
-
pEXPR: hEF1a-TAL21<br>
+
-
DOX Induction<br>
+
-
L7ae tuning experiment<br>
+
-
Transfect<br>
+
-
TAL14 tuning experiment<br>
+
-
NOTES<br>
+
-
hEF1a-TAL21 had a red pellet during midi prep spin down --> re-LR & re pick colonies<br>
+
-
<br>
+
-
8.1<br>
+
-
Keep updating the parts tree (include experiments)<br>
+
-
Double input low sensor??<br>
+
-
Where are the siRNA?<br>
+
-
Mini prep<br>
+
-
miR-30d (TAL14)<br>
+
-
miR-16a (TAL14)<br>
+
-
let-7f (TAL14)<br>
+
-
pEXPR: hEF1a-TAL21<br>
+
-
Send for sequencing<br>
+
-
miR-30d (TAL14)<br>
+
-
miR-16a (TAL14)<br>
+
-
let-7f (TAL14)<br>
+
-
Digest and Run gel<br>
+
-
MAV1212-hEF1a-TAL21<br>
+
-
Flow Cytometry<br>
+
-
L7ae tuning experiment<br>
+
-
Transform <br>
+
-
miR-125b (TAL14)<br>
+
-
NOTES<br>
+
-
minipreps took a really long time --> TAL21 wasn't digested<br>
+
-
siRNA should be coming "soon"<br>
+
-
since HS_125b didn't grow colonies --> re transformed<br>
+
-
<br>
+
-
8.4<br>
+
-
Keep updating the parts tree (include experiments)<br>
+
-
miRNA Sensors (Theory)<br>
+
-
Meet with Kyle about quantifying repression<br>
+
-
Troubleshoot gel results<br>
+
-
Digest and run gel<br>
+
-
MAV1212-hEF1a-TAL14<br>
+
-
MAV1212-hEF1a-TAL21<br>
+
-
LR (if digest fails)<br>
+
-
MAV1212-hEF1a-TAL21 - find pENTR TAL21<br>
+
-
Pick colonies (if digest failes)<br>
+
-
pENTR TAL14<br>
+
-
double input low sensor<br>
+
-
<br>
+
-
8.7<br>
+
-
Keep updating the parts tree (include experiments)<br>
+
-
Meet with Kyle about quantifying repression<br>
+
-
Miniprep<br>
+
-
pENTR TAL14<br>
+
-
Double-Input Low Sensor<br>
+
-
Find<br>
+
-
pEXPR hEF1A:TAL14<br>
+
-
pENTR TAL21<br>
+
-
pEXPR hEF1A:TAL21<br>
+
-
LR <br>
+
-
MAV1212-hEF1a-TAL14<br>
+
-
MAV1212-hEF1a-TAL21<br>
+
-
Sequence<br>
+
-
Double-input low sensor<br>
+
-
Digest & run gel<br>
+
-
pENTR: TAL14<br>
+
-
Transfect<br>
+
-
L7ae quantification experiment<br>
+
-
NOTES<br>
+
-
<br>
+
-
8.8<br>
+
-
Keep updating the parts tree (include experiments)<br>
+
-
Figure out MATLAB quantification<br>
+
-
Make plate maps for siRNA transfection<br>
+
-
Analyze Digest<br>
+
-
pENTR TAL14<br>
+
-
Analyze Sequencing Results<br>
+
-
Double-Input Low Sensor<br>
+
-
Add DOX<br>
+
-
L7ae quantification experiment<br>
+
-
LR <br>
+
-
hEF1a-TAL14 (if digest works)<br>
+
-
hEF1a-TAL21 (find entry vector / plate)<br>
+
-
NOTES<br>
+
-
<br>
+
-
8.11<br>
+
-
Keep updating the parts tree (include experiments)<br>
+
-
FIND pENTR: TAL21 <br>
+
-
TASBE tools with Brian<br>
+
-
Anaylze sequencing results<br>
+
-
Pick up sensors from Jeremy<br>
+
-
Clean up experiments page<br>
+
-
Transform<br>
+
-
hEF1a-TAL14 (LR)<br>
+
-
Sensors from Jeremy? <br>
+
-
Inoculate midi culture<br>
+
-
Double input low sensor<br>
+
-
Tissue Culture<br>
+
-
Seed plate for miRNA sensors w/o siRNA<br>
+
-
Check dilutions in TC room<br>
+
-
NOTES<br>
+
-
<br>
+
-
8.12<br>
+
-
Keep updating the parts tree (include experiments)<br>
+
-
FIND pENTR: TAL21 <br>
+
-
TASBE tools with Brian<br>
+
-
Pick up sensors from Jeremy<br>
+
-
Tissue Culture<br>
+
-
Seed plate for miRNA sensors w/ siRNA??<br>
+
-
Check dilutions in TC room<br>
+
-
NOTES<br>
+
-
<br>
+
-
8.13<br>
+
-
Keep updating the parts tree (include experiments)<br>
+
-
Emal Jake Beal about TASBE tools<br>
+
-
Pick up sensors from Jeremy<br>
+
-
Tissue Culture<br>
+
-
Transfect plate for miRNA sensors w/ siRNA<br>
+
-
Miniprep<br>
+
-
hEF1a-TAL14<br>
+
-
(Restriction Digest and Run Gel)<br>
+
-
hEF1a-TAL14<br>
+
-
(Golden Gate)<br>
+
-
TAL14 High Sensors<br>
+
-
NOTES<br>
+
-
<br>
+
-
8.14<br>
+
-
Keep updating the parts tree (include experiments)<br>
+
-
Emal Jake Beal about TASBE tools<br>
+
-
Pick up sensors from Jeremy<br>
+
-
And figure out what/when to do with them...<br>
+
-
New map for siRNA experiments<br>
+
-
Re-name siRNAs as "siRNA-[insert number here]"<br>
+
-
Figure out scatterplot in MATLAB<br>
+
-
Tissue Culture<br>
+
-
Cytometry for miRNA sensors w/o siRNA<br>
+
-
Golden Gate<br>
+
-
TAL14 High Sensors<br>
+
-
Grow in midi culture<br>
+
-
Double input low sensor<br>
+
-
GG Donor for TC<br>
+
-
hEF1a-TAL21<br>
+
-
<br>
+
-
8.15<br>
+
-
Keep updating the parts tree (include experiments)<br>
+
-
Meet with Kyle about scatterplots in MATLAB<br>
+
-
Tissue Culture<br>
+
-
Cytometry for miRNA sensors with siRNA<br>
+
-
Digest verify<br>
+
-
MAV1212 hEF1a:Tal14<br>
+
-
Golden Gate<br>
+
-
TAL14 High Sensors<br>
+
-
Midi Prep<br>
+
-
Double input low sensor<br>
+
-
GG Donor for TC<br>
+
-
NOTES<br>
+
-
hEF1a-TAL14 is still giving us troubles (the gel doesn't quite make sense)
+
-
<h2>Native Receptor</h2>
+
<tr><td><h3 align="center" style="font-size:42px; color:teal"><b> NOTEBOOKS</b></h3><br></td></tr>
-
<br>
+
<tr><td><p style="font-size:12px" align=center><i>Attributions: James Anderson (Treatment), Gary Burnett (miRNA), <br> Shinjini Saha (Native Receptor), Alex Smith (B-Cell Receptor)</i></p></td></tr>
-
<br>June 15
+
<tr><td align=center> <img src="https://static.igem.org/mediawiki/2014/7/76/MIT_2014_Notebook_icon.png"> </td></tr>
-
<br>AG, LA SS
+
</table>
-
<br>Grew pENTR_LilrB2, pENTR_PirB
+
<br><br><br>
-
<br>
+
<center><b>
-
<br>June 16
+
<h2><a href="https://2014.igem.org/Wiki/2014.igem.org/Team:MIT/native_receptor_notebook" style="color:black">NATIVE RECEPTOR LAB NOTEBOOK</h2></a>
-
<br>Liquid cultures didn't grow
+
<h2><a href="https://2014.igem.org/Wiki/2014.igem.org/Team:MIT/BCR_notebook" style="color:black">B-CELL RECEPTOR LAB NOTEBOOK</h2></a>
-
<br>SS
+
<h2><a href="https://2014.igem.org/Wiki/2014.igem.org/Team:MIT/miRNA_notebook" style="color:black">miRNA DETECTION LAB NOTEBOOK</h2></a>
-
<br>New liquid cultures made
+
<h2><a href="https://2014.igem.org/Wiki/2014.igem.org/Team:MIT/Treatment_notebook" style="color:black">TREATMENT LAB NOTEBOOK</h2></a>
-
<br>AG, LA
+
</b></center>
-
<br>NEGEM and High School presentations completed
+
<br><br><br>
-
<br>
+
-
<br>June 17
+
-
<br>Liquid culture still didn't grow BUT we figured out why
+
-
<br>SS
+
-
<br>Made new cultures and incubate them overnight
+
-
<br>Made ggplates
+
-
<br>LA
+
-
<br>Prepare for a practice HEK293 transfection with Nelson
+
-
<br>AG
+
-
<br>Send ggDONR for sequencing
+
-
<br>Split HEK293 cells
+
-
<br>
+
-
<br>June 18
+
-
<br>Liquid culture STILL didn't grow
+
-
<br>New plates + old ggDONR grows and turn blue
+
-
<br>We think something is wrong with the plates
+
-
<br>Primers for fusion have arrived
+
-
<br>LA
+
-
<br>Prepared media for HEK293 in preparation for transfection transfection
+
-
<br>
+
-
<br>June 19
+
-
<br>Plates from our transformation grew blue and white colonies
+
-
<br>PirB2 didn't grow any white colonies
+
-
<br>SS
+
-
<br>Liquid culture - to make stock - of ggDONR from troubleshooting experiment
+
-
<br>golden gated again
+
-
<br>AG
+
-
<br>Made liquid culture of GGDonr and pENTR_L1_LilrB2_L2 for miniprep
+
-
<br>
+
-
<br>June 20
+
-
<br>SS
+
-
<br>Took liquid cultures out of incubator and put in fridge.
+
-
<br>AG, LA, SS
+
-
<br>Attended NEGEM 3.1
+
-
<br>
+
-
<br>June 22
+
-
<br>LA
+
-
<br>Transformed GoldenGated pENTR_L1_LilrB2_L2, pENTR_L1_PirB_L2
+
-
<br>Plated
+
-
<br>SS
+
-
<br>Made cell stock of GGDonr and pENTR_L1_LilrB2_L2 (from previous GoldenGate)
+
-
<br>Miniprepped GGDonr and pENTR_L1_LilrB2_L2 (from previous GoldenGate)
+
-
<br>AG
+
-
<br>Problem with HEK-293 cell culture - culture another plate
+
-
<br>
+
-
<br>June 23
+
-
<br>AG
+
-
<br>Transformed Golden Gate product:
+
-
<br>positive control didn't grow any colonies
+
-
<br>blue and white colonies on LilrB2
+
-
<br>only white colonies on PirB
+
-
<br>picked colonies
+
-
<br>SS
+
-
<br>Digested previous pENTR_LilrB2 and GGDonr (BssSI, 3.1)
+
-
<br>Could not figure out how to make the gel imaging station to work. But...
+
-
<br>Gel did not fluoresce under UV, not even the hyperladder.
+
-
<br>
+
-
<br>June 24
+
-
<br>Liquid cultures from the second golden gate grew
+
-
<br>SS
+
-
<br>Liquid cultures miniprepped and DNA waiting to be verified
+
-
<br>
+
-
<br>June 25
+
-
<br>AG
+
-
<br>Ran verification gels of the products of the minipreps - one very bright band for each lane
+
-
<br>We suspect enzyme (BssSI) didn't work - DNA was super coiled. Old enzyme has been thrown out.
+
-
<br>Split HEK293 cells. Prepare for practice transfection
+
-
<br>
+
-
<br>June 26
+
-
<br>SS
+
-
<br>Digested LilrB2, PirB and GGDonr (again). EcoRI this time
+
-
<br>AG
+
-
<br>Practice transfection (fluorescent protein - 1 plasmid)
+
-
<br>
+
-
<br>June 29
+
-
<br>AG
+
-
<br>Plated 3rd GG pENTR_LilrB2, pENTR_Pirb (Treatment group did control.)
+
-
<br>Flow cytometry of practice transfection - high efficiency
+
-
<br>Prepare for more complex transfection - 2 plasmids
+
-
<br>
+
-
June 30
 
-
<br>LA
 
-
<br>Picked colonies
 
-
<br>cultures grew
 
-
<br>found inactive cofilin plasmid
 
-
<br>need to verify sequence and see if it has been used before
 
-
<br>if suitable - will order today
 
-
<br>started designing rest of fusions on geneious
 
-
<br>AG
 
-
<br>Transfection - 2 plasmids
 
-
<br>
 
-
<br>July 1
 
-
<br>SS
 
-
<br>Miniprepped DNA
 
-
<br>Restriction digested for verification
 
-
<br>cofilin order on the plasmid page
 
-
<br>
 
-
<br>July 2
 
-
<br>SS
 
-
<br>Ran gels of yesterday's digest
 
-
<br>Did digests again on select pENTRs
 
-
<br>40ul total volume instead of 20ul, same amount DNA (500ng)
 
-
<br>Ran gel
 
-
<br>AG
 
-
<br>Flow cytometry - lower efficiencies (keep practicing).
 
-
<br>Train team members in tissue culture
 
-
<br>
 
-
<br>July 3-2
 
-
<br>SS
 
-
<br>Imaged gel that we ran yesterday
 
-
<br>
 
-
<br>July 6
 
-
<br>SS
 
-
<br>Transformed LR Product (pEXPR hEF1a:PirB and hEF1a:LilrB2)
 
-
<br>Picked colonies (3 each) and grew up overnight in liquid culture
 
-
<br>Mini-prepped and nano-dropped
 
-
<br>AG
 
-
<br>Split HEK293 cells
 
-
<br>
 
-
<br>July 7
 
-
<br>LA
 
-
<br>PCRs to make fusions started
 
-
<br>50 ml liquid cultures for midi prep in incubator
 
-
<br>
 
-
<br>July 8
 
-
<br>AG
 
-
<br>pEXPRs midiprepped
 
-
<br>LR product digested and gel ran
 
-
<br>LA
 
-
<br>PCRs for fusions in progress
 
-
<br>Digesting LR's
 
-
<br>Hef1a:LirB2 BsrGI
 
-
<br>Hef1a:PirB ClaI
 
-
<br>
 
-
<br>July 9
 
-
<br>Sequencing results received for pENTR
 
-
<br>LilrB2 is correct
 
-
<br>PirB: Qsite ligation with the C terminus gblock in pENTR
 
-
<br>New PirB colonies picked
 
-
<br>Restriction digest on pEXPR LilrB2 repeated because we suspected quality of digestion
 
-
<br>LilrB2 = 1900 bps
 
-
<br>PirB = 2500 bps (won't work because pENTR is not what we thought it was)
 
-
<br>AG
 
-
<br>Split HEK293 cells. Start 3 plate series so that splitting would occur every day.
 
-
<br>
 
-
<br>July 10
 
-
<br>SS
 
-
<br>Digested pENTR_PirB with EcoRI.
 
-
<br>Made primers for PirB, LilrB2, Cofilin fusion to YFP.
 
-
<br>LA
 
-
<br>Sent pENTR_PirB for sequencing for M13 F and R primers.
 
-
<br>Sent pEXPR_hEF:LilrB2 from yesterday that seemed to work (L2, L3) for full sequencing.
 
-
<br>
 
-
<br>July 11
 
-
<br>LA
 
-
<br>Send pEXPR hEF1a:LilrB2 and pENTR PirBfor sequencing (L2, L3)
 
-
<br>
 
-
<br>July 14
 
-
<br>AG
 
-
<br>TEVp-cofilin fusion to be ordered as a gBlock
 
-
<br>Split HEK293 cells.
 
-
<br>LA
 
-
<br>Sequencing results returned and analyzed
 
-
<br>pEXPR hEF1a:LilrB2 didn't give us a good enough read - we're sending those out again
 
-
<br>pENTR L1_PirB_L2 worked
 
-
<br>SS
 
-
<br>L1_PirB_L2 LRed
 
-
<br>
 
-
<br>July 16
 
-
<br>SS
 
-
<br>Received primers - started PCRs
 
-
<br>Done:
 
-
<br>LilrB2 (eYFP)
 
-
<br>PirB (eYFP)
 
-
<br>LilrB2 (TCS-Gal4VP16)
 
-
<br>PirB (TCS-Gal4VP16)
 
-
<br>To be done:
 
-
<br>Cofilin (eYFP)
 
-
<br>Ran verification gel on PCR products
 
-
<br>
 
-
<br>LA
 
-
<br>streaked cofilin plasmid DNA
 
-
<br>Sequencing results returned for LilrB2 pEXPR
 
-
<br>LilrB2 insert is there: LilrB2 pEXPR verified
 
-
<br>"No Priming" for second read in both samples?
 
-
<br>AG
 
-
<br>PirB pEXPR transformed
 
-
<br>Split HEK293 cells.
 
-
<br>
 
-
<br>July 17
 
-
<br>SS
 
-
<br>PirB LR colonies picked and liquid cultures prepared
 
-
<br>LilrB2 PCRs repeated under 2 different (higher) annealing temperatures
 
-
<br>Remaining PirB PCR product ran and band with the correct size DNA to be extracted
 
-
<br>Gel with PCR products imaged
 
-
<br>Expected sizes:
 
-
<br>LilrB2: 2,000
 
-
<br>PirB: 2,500
 
-
<br>
 
-
<br>July 18
 
-
<br>SS
 
-
<br>PirB PCR bands extracted
 
-
<br>Concentrations very low
 
-
<br>around 5 ng/ul
 
-
<br>PirB pEXPRs miniprepped
 
-
<br>Restriction digest on PirB pEXPRs run
 
-
<br>LA
 
-
<br>PirB PCRs run again
 
-
<br>LilrB2 PCRs done at higher annealing temperatures
 
-
<br>Cofilin plasmid colonies picked
 
-
<br>AG
 
-
<br>Tissue culture
 
-
<br>
 
-
<br>July 19
 
-
<br>pEXRP_hEF1a:PirB 2 seems to give the correct band pattern,
 
-
<br>but the bands are slightly lower than they should be.
 
-
<br>Ran extraction gel for PCR products: HL1. LY65, LT67, LY68, LT70, PY, PT
 
-
<br>Turns out the gel was upside down, so all the products diffused. Need to do all the PCR's again
 
-
<br>SS
 
-
<br>Cofilin plasmid miniprepped. Concentrations ~600ng/ul
 
-
<br>Re-did PirB PCR's, Did Cofilin PCR. LilrB2 PCR's need to be redone
 
-
<br>Current cell glycerol stocks: pENTR_PirB (verified), pENTR_LilrB2 (verified), pEXPR_hEF1a:LilrB2 (verified), <br>pEXPR_hEF1a: PirB (unverified), Cofilin gene in backbone.
 
-
<br>AG
 
-
<br>Tissue culture
 
-
<br>
 
-
<br>July 20
 
-
<br>SS
 
-
<br>Did PCR's of LilrB2
 
-
<br>Ran gel of all the PCR's
 
-
<br>Ladder, LilrB2 YFP 65, LilrB2 YFP 68, LilrB2 TCSG4 67, LilrB2 TCSG4 70, PirB YFP, PirB TCSG4, Cofilin YFP
 
-
<br>
 
-
<br>July 21
 
-
<br>SS
 
-
<br>Miniprepped new pEXPR_hEF1a: PirB (4-8) ~200ng/uL
 
-
<br>Digested new pEXPR_hEF1a: PirB
 
-
<br>Ran Gel
 
-
<br>Looks like they all worked!
 
-
<br>Sent pEXPR_hEF1a: PirB 2 (from saturday) for sequencing. Will not be good quality (did not have enough DNA)
 
-
<br>Transform pEXPR_hEF1a: PirB (4-8)
 
-
<br>Plate pEXPR_hEF1a:LilrB2 (cell stock)
 
-
<br>AG
 
-
<br>Tissue culture
 
-
<br>
 
-
<br>July 22
 
-
<br>SS
 
-
<br>Liquid culture for hEF1a:(Receptor) midiprep grown up - to be midiprepped tomorrow
 
-
<br>eYFP-cofilin golden gate transformed
 
-
<br>
 
-
<br>July 23
 
-
<br>SS
 
-
<br>Miniprep pEXPR_hEF1a: LilrB2/PirB:
 
-
<br>A couple came out to have around 700ng/ul. Others were around 400ng/ul
 
-
<br>Midiprep Culture:
 
-
<br>Picked 2 out of 5 pEXPR_hEF1a: PirB's to inoculate midiprep cultures, but kept the miniprep cultures of the other 3 just in case.
 
-
<br>Also inoculated pEXPR_hEF1a: LilrB2.
 
-
<br>Midiprep party tomorrow!
 
-
<br>eYFP-Cofilin:
 
-
<br>Plate had lots of white colonies and a few blue ones. Not expected. Expecting a lot of backbone re-ligation. Picked 10. <br>Will miniprep tomorrow.
 
-
<br>AG
 
-
<br>Tissue culture
 
-
<br>
 
-
July 24
 
-
<br>SS
 
-
<br>Miniprepped eYFP:Cofilin colonies
 
-
<br>Ran digest gel (PstI, ApaI, Cutsmart)
 
-
<br>2 might have worked
 
-
<br>Sent for sequencing.
 
-
<br>Turns out, didn't work. eYFP missing.
 
-
<br>
 
-
<br>July 25
 
-
<br>SS
 
-
<br>Did midipreps. Very strange distribution of concentrations:
 
-
<br>LilrB2 1: ~1200
 
-
<br>LilrB2 2: ~700
 
-
<br>PirB 7: ~4000
 
-
<br>PirB8: ~300
 
-
<br>Digesting midipreps just in case.
 
-
<br>LilrB2: XbaI, ApaI
 
-
<br>PirB: ApaI
 
-
<br>
 
-
<br>LilrB2's are weird. Running undigested controls.
 
-
<br>LirB2 DNA is contaminated!
 
-
<br>Correct band pattern is most definitely there. Which means all parts: hEF1a promoter, LilrB2 gene and backbone are <br>there.
 
-
<br>AG
 
-
<br>Tissue culture
 
-
<br>July 26
 
-
<br>SS
 
-
<br>Run DNA for gel extraction. Also look to see if other, verified DNA contaminated or not.
 
-
<br>Pick colonies fusions
 
-
<br>Transform
 
-
<br>Transforming with L2 and L3 DNA (L24, L34).
 
-
<br>
 
-
<br>
 
-
July 27
 
-
<br>SS
 
-
<br>Minipreped a gazillion fusions.
 
-
<br>4/5 cofilins seemed to work. Had 3 fragments. Means at least 2 of the goldengated parts are present. Will digest <br>with ApaI tomorrow to check if 3rd is present.
 
-
<br>PirBs didn't seem to work, will pick more colonies.
 
-
<br>LilrB2 didn't work
 
-
<br>Will pick pEXPR_LilrB2 colonies.
 
-
<br>AG
 
-
<br>Tissue culture
 
-
 
-
<br>July 28
 
-
<br>SS
 
-
<br>Cofilin 1, 3, 4, 5 worked, need to do another digest.
 
-
<br>LilrB2:TCSG4 1, 4, 5 might have worked. Strange extra band on top. Will do different digest.
 
-
<br>LilrB2: YFPL none worked. Will miniprep more.
 
-
<br>PirB:TCSG4 none worked.
 
-
<br>PirB:YFP none worked.
 
-
<br>Cofilin worked! Sending all 4 for sequencing, 'cuz PCRs and mutations, ya know?
 
-
<br>LilrB2:TCSG4 did not work. Will have to miniprep more.
 
-
<br>
 
-
<br>Made glycerol cell stocks of all the cofilins, but due to some mistake, the labels got erased. So, unless they all <br>worked, I'll just re-transform one that worked.
 
-
<br>Making glycerol cell stocks of pEXPR_hEF1a: LilrB2 (L2 and L3)
 
-
<br>
 
-
<br>July 29
 
-
<br>midipreped pEXPR_hEF1a:LilrB2.
 
-
<br>All the eYFP:Cofilin worked! LRed today.
 
-
<br>Did more golden gates.
 
-
<br>Picked more colonies, too.
 
-
<br>Inoculated midi culture for ggDonr
 
-
<br>
 
-
<br>July 30
 
-
<br>More fusions digested!
 
-
<br>New goldengates of fusion proteins transformed
 
-
<br>New LR of hEF1a:YFPCofilin transformed
 
-
<br>LR of TREt:YFPCofilin done.
 
-
<br>
 
-
<br>July 31
 
-
<br>SS
 
-
<br>LTs might have worked. Did digest again with different enzymes.
 
-
<br>Did digest with some other interesting ones: LT12 and PT11 and PT12.
 
-
<br>Transformed TREt:TEVpCofilin LRs
 
-
<br>Picked hEF1a:TEVpCofilin LRs
 
-
<br>Picked pENTR:YFPCofilin
 
-
<br>Picked more LY and PY from new goldengates
 
-
<br>Sent LT and PT for sequencing
 
-
<br>
 
-
<br>LA
 
-
<br>Transfected LilrB2 for membrane localization
 
-
<br>Gel verification of LilrB2 and PirB PCR products ran
 
-
<br>
 
-
<br>August 1
 
-
<br>SS
 
-
<br>Miniprep PY, LY, pENTR_TEVpCofilin
 
-
<br>Verify Minipreps
 
-
<br>Transform hEF1a:YFPCofilin
 
-
<br>Pick TREt:YFPCofilin Colonies
 
-
<br>All 4 pENTR_TEVpCofilin worked!
 
-
<br>The others didn't work. Given the lack of blue colonies, I'm thinking backbone religation/only one part present <br>(probably YFP). Need to figure out something else.
 
-
 
-
<br>Sequencing:
 
-
<br>LilrB2TCSG4 seems to have worked! 11 and 13 might have point mutations (high G content, unlikely). Will proceed with <br>12.
 
-
<br>PirBTCSG4 seems to have worked, too! 13 seems to have a point mutation (high G content, unlikely). Will proceed with <br>11.
 
-
<br>
 
-
<br>August 2
 
-
<br>SS
 
-
<br>Transform all LRs (hEF1a:PT, hEF1a:LT, hEF1a:TC, TREt:TC)
 
-
<br>Pick hEF1a:YC colonies
 
-
<br>Miniprep TREt:YC if it worked. Otherwise, wait for Brian. Worked.
 
-
<br>Midiprep inoculate TREt:YC
 
-
<br>Digest LilrB2TCSG4 with NcoI and PstI seperately.
 
-
<br>Digest TREt:YC with ApaI
 
-
<br>
 
-
<br>TREt:YFP_Cofilin 1, 2, 4 verified. Will midiprep 2.
 
-
<br>Ran PT (pENTR_PirBTCSG4) to make sure it isn't contaminated).
 
-
<br>The gel for PT had an extra band, but the sequencing results show that the TCSG4 is present and goes into the PirB <br>region. That means everything is in order.
 
-
<br>
 
-
 
-
<br>August 3
 
-
<br>SS
 
-
<br>Took pEXPR TREt YC midiprep out of incubator
 
-
<br>Took pEXPR_hEF1a:YC miniprep out of incubator
 
-
<br>Took plates out of the incubator
 
-
<br>plates dried out. Picked colonies for TREt-TC
 
-
<br>
 
-
<br>August 4
 
-
<br>SS
 
-
<br>Miniprep pEXPRs (TREt TC, hEF1a YC)
 
-
<br>Check pEXPRs (TREt TC, hEF1a YC)
 
-
<br>Inoculate Midipreps
 
-
<br>Pick colonies for hEF1a LT, PT, TC
 
-
<br>Digest TREtTC with ApaI Cutsmart
 
-
<br>Digest hEF1aYC with BsrGI 2.1
 
-
<br>Digest LT with BglI 3.1
 
-
<br>Digest TC with PvuII with 3.1
 
-
<br>Digest LT11 with BlpI with CS
 
-
<br>Give up on hEF1aYC. Won't be using it anyway.
 
-
<br>
 
-
<br>pEXPR_TREt:TEVpCofilin is wrong. Gives weird patterns. Need to pick again.
 
-
<br>pENTR:LilrB2TCSG4 is wrong. Big chunk is actually missing. Need to pick again.
 
-
<br>
 
-
<br>August 5
 
-
<br>SS
 
-
<br>Miniprep pEXPR (hEF1a: PirBTCS, hEF1a: TEVpCofilin)
 
-
<br>Verify hEF1a: PirBTCS
 
-
<br>Miniprep pENTR LilrB2TCS
 
-
<br>Verify pENTR LilrB2TCS
 
-
<br>Midiprep inoculate hEF1a: PirBTCS
 
-
<br>Pick more TREt:TEVpCofilin
 
-
<br>Plate more TREt:TEVpCofilin if possible.
 
-
 
-
<br>Digest pEXPR_hEF1a: PirBTCSG4VP16 with BsrGI, buffer 2.1
 
-
<br>Digest pENTR_LilrB2:TCSG4VP16 with XhoI +XbaI, buffer CS
 
-
<br>Order: hEF1a PT 1, 2, 3, 4, LT16, 17, 18, 19
 
-
 
-
<br>August 6
 
-
<br>SS
 
-
<br>Miniprepped more pENTR_LilrB2TCS
 
-
<br>Verify hEF1a: PirBTCS. Else pick more colonies. Picked more colonies
 
-
<br>Verify pENTR LilrB2TCS. Including LT16. Did not work
 
-
 
-
<br>Verify pEXPR_TREt:TEVpCofilin. Worked!
 
-
 
-
<br>Midiprep inoculate TREt:TEVpCofilin if it worked.
 
-
<br>Midiprep inoculate hEF1a: PirBTCS if it worked. Didn't work
 
-
<br>Sent pENTR_LilrB2 (04 2) for sequencing again...
 
-
 
-
<br>Digest pEXPR_hEF1a: PirBTCS (1, 3, 4) with BsrGI, 2.1
 
-
<br>Digest LT (21-25) with XbaI, XhoI, CS
 
-
<br>Digest LT16 with NcoI, XhoI, CS
 
-
<br>Digest pEXPR_TREt:TEVpCofilin with BsrGI, 2.1
 
-
 
-
<br>hEF1a: PirBTCS did not work. Need to pick more
 
-
 
-
<br>
 
-
<br>August 7
 
-
<br>SS
 
-
<br>Miniprepped more hEF1a: PirBTCSG4. Some worked!
 
-
<br>Midiprep inoculating.
 
-
<br>Also cell stock
 
-
<br>PCR'd LilrB2 and YFP. LilrB2 band is too low. YFP didn't work.
 
-
<br>Midiprepped TREt:YFPCofilin and TREt:TEVpCofilin.
 
-
 
-
<br>
 
-
<br>August 8
 
-
<br>SS
 
-
<br>Midiprep pEXPR_PirBTCSG4VP16. The midiprep concentration was low, around 16. Probably because the culture didn't grow <br>very well.
 
-
<br>Miniprep inoculating again.
 
-
<br>Digest any YFP fusion minipreps we have to check for what segments are present and what went wrong.
 
-
<br>Dilute midipreps.
 
-
 
-
<br>
 
-
<br>August 12
 
-
<br>AG
 
-
<br>hEF1a:PirB-TCS-Gal4VP16 midiprepped
 
-
<br>LilrB2 pENTR sent for sequencing
 
-
<br>
 
-
<br>August 14
 
-
<br>AG
 
-
<br>pENTR LilrB2:
 
-
<br>Blunt end cloning of gBlock
 
-
<br>
 
-
<br>pEXPR hEF1a:PirB-YFP
 
-
<br>Transform
 
-
<br>
 
-
<br>
 
-
<br>August 22
 
-
<br>AG
 
-
<br>Miniprepped pENTR-LilrB2 (1-6)
 
-
<br>Ran digests.
 
-
<br>They look good.
 
-
<br>Sent LilrB2 1 for full sequencing. Waiting till sequence verified for making cell stock.
 
-
<br>
 
-
<br>August 23
 
-
<br>SS
 
-
<br>Transformed pEXPR_hEF1a:LilrB2 and pEXPR_TREt:LilrB2
 
-
<br>
 
-
<br>August 24
 
-
<br>SS
 
-
<br>Got sequencing results back. The pENTR is sequence verified!
 
-
<br>Picked colonies for pEXPRs.
 
-
<br>
 
-
<br>August 25
 
-
<br>Miniprep LRs
 
-
<br>Digest LRs
 
-
<br>Also digest pEXPR_hEF1a: PirBYFP
 
-
<br>PCRing at 53 degress, 40 seconds.
 
-
<br>Digesting with:
 
-
<br>pEXPR_hEF1a: LilrB2 XhoI, CS
 
-
<br>pEXPR_TREt: LilrB2 XbaI, CS
 
-
<br>pEXPR_hEF1a: PirB_YFP ApaI, CS
 
-
<br>
 
-
<br>Both LilrB2 pEXPRs worked!
 
-
<br>
 
-
<br>August 26
 
-
<br>SS
 
-
<br>Midis didn't work. Had ~10ng/ul concentration.
 
-
<br>So, taking the cell stocks and throwing them out.
 
-
<br>
 
-
<br>August 27
 
-
<br>SS
 
-
<br>The pENTR_PirB_YFP did not actually have the YFP insert.
 
-
<br>
 
-
<br>August 28
 
-
<br>SS
 
-
<br>Picked pENTR LilrB2_YFP/TCS colonies
 
-
<br>Put pEXPR_LilrB2 midi culture in incubator (~8:45pm)
 
-
<br>
 
-
<br>August 31
 
-
<br>SS
 
-
<br>Transformed pEXPR hEF1a:LilrB2 and TREt:LilrB2 (10pm)
 
-
<br>Digesting pENTR LilrB2YFP and LilrB2TCS
 
-
<br>LilrB2YFP: PstI                  3.1
 
-
<br>LilrB2TCS: XhoI + XbaI    CS
 
-
<br>
 
-
<br>Looks like LT4 worked. Will send for sequencing (M13F and R)
 
-
<br>The LYs don't seem to have worked. May be a problem with the YFP PCRs.
 
-
<br>
 
-
<br>September 1
 
-
<br>SS
 
-
<br>Inoculated midi for pEXPR(hEF1a/TREt):LilrB2
 
-
<br>
 
-
<br>September 2
 
-
<br>Midiprepped pEXPR(hEF1a/TREt):LilrB2
 
-
<br>Concentrations around 1ng/ul.
 
-
<br>pENTR_LilrB2TCS sequencing results came. Both components are there.
 
-
<br>
 
-
<br>September 3
 
-
<br>SS
 
-
<br>LR'd LilrB2TCS (hef1a, tret)
 
-
<br>
 
-
<br>September 4
 
-
<br>SS
 
-
<br>Transformed LRs
 
-
<br>Tried midiprepping pEXPR_hef1a/tret:LilrB2. The vacuum manifold wasn't working.
 
 +
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Latest revision as of 03:48, 18 October 2014

 


Image Map


NOTEBOOKS


Attributions: James Anderson (Treatment), Gary Burnett (miRNA),
Shinjini Saha (Native Receptor), Alex Smith (B-Cell Receptor)




NATIVE RECEPTOR LAB NOTEBOOK

B-CELL RECEPTOR LAB NOTEBOOK

miRNA DETECTION LAB NOTEBOOK

TREATMENT LAB NOTEBOOK