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| | <head></head> | | <head></head> |
| | <body> | | <body> |
| - | | + | <div style="padding:20px"> |
| | <table width="90%" align="center"> | | <table width="90%" align="center"> |
| | | | |
| - | <tr><td><h3 align="center" style="font-size:45px">Lab Notebooks</h3><br></td></tr> | + | <tr><td><h3 align="center" style="font-size:42px; color:teal"><b> NOTEBOOKS</b></h3><br></td></tr> |
| | + | <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> |
| | <tr><td align=center> <img src="https://static.igem.org/mediawiki/2014/7/76/MIT_2014_Notebook_icon.png"> </td></tr> | | <tr><td align=center> <img src="https://static.igem.org/mediawiki/2014/7/76/MIT_2014_Notebook_icon.png"> </td></tr> |
| | </table> | | </table> |
| | + | <br><br><br> |
| | + | <center><b> |
| | + | <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> |
| | + | <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> |
| | + | <h2><a href="https://2014.igem.org/Wiki/2014.igem.org/Team:MIT/miRNA_notebook" style="color:black">miRNA DETECTION LAB NOTEBOOK</h2></a> |
| | + | <h2><a href="https://2014.igem.org/Wiki/2014.igem.org/Team:MIT/Treatment_notebook" style="color:black">TREATMENT LAB NOTEBOOK</h2></a> |
| | + | </b></center> |
| | + | <br><br><br> |
| | | | |
| | | | |
| - | <h2>miRNA</h2>
| |
| - | 6.22<br>
| |
| - | Update the parts list on the wiki<br>
| |
| - | Create an experiments page on the wiki<br>
| |
| - | Prepare to meet with Jake Beal<br>
| |
| - | Re-transform MAV1212<br>
| |
| - | Re-transform MAV1212-Hef1a-eBFP2<br>
| |
| - | Mini prep MAV1212-Hef1a-eBFP2<br>
| |
| - | 6.23<br>
| |
| - | Go over workflow of plasmid construction as a group<br>
| |
| - | Finish a unified daily log<br>
| |
| - | Digest meeting with Jake Beal<br>
| |
| - | Go through plates<br>
| |
| - | Re plate MAV1212<br>
| |
| - | Pick colonies for MAV1212<br>
| |
| - | Plan LacZ PCR product<br>
| |
| - | Primers ( ? )<br>
| |
| - | Figure out what's going on<br>
| |
| - | <br>
| |
| - | 6.24<br>
| |
| - | Present about cell models<br>
| |
| - | COMPLETE PARTS LIST ON WIKI<br>
| |
| - | Prep experiments<br>
| |
| - | Contact Jeremy about multiple miRNA target sites chained together<br>
| |
| - | Pick colonies for MAV1212<br>
| |
| - | Miniprep MAV1212<br>
| |
| - | Keep MAV1212-Hef1a-eBFP2 (for use WITHOUT target sites)<br>
| |
| - | Gateway MAV1212-Hef1a-L7ae (for high sensor)<br>
| |
| - | Gateway MAV1212_Hef1a-eYFP (for use WITH target sites)<br>
| |
| - | Transform Hef1a, MAV1212 ( ? )<br>
| |
| - | NOTES:<br>
| |
| - | No MAV1212 colonies grew on the plates or in the medium<br>
| |
| - | Jeremy gave us more, enough for one more transformation<br>
| |
| - | 6.25<br>
| |
| - | Create a clear workflow<br>
| |
| - | Update "status" of plasmids on parts list<br>
| |
| - | How do we string multiple low sensors together?<br>
| |
| - | Determine which pairs of target sites we want<br>
| |
| - | Find sequences (hopefully under 200 bp)<br>
| |
| - | Send ultramer order to Brian<br>
| |
| - | Tranform LRs <br>
| |
| - | MAV1212-Hef1a-mKate2<br>
| |
| - | MAV1212-Hef1a-L7ae<br>
| |
| - | Hef1a on Kan<br>
| |
| - | Pick colonies from transformations<br>
| |
| - | MAV1212-Hef1a-eBFP2<br>
| |
| - | Golden Gate<br>
| |
| - | Gather miRNA target sites from Jeremy<br>
| |
| - | Make all 6 single low sensors<br>
| |
| - | NOTES<br>
| |
| - | MAV1212 didn't grow (ccdB is over expressed)<br>
| |
| - | Hef1a was plated on Amp plates...but it has Kan resistance<br>
| |
| - | Verify MAV1212-Hef1a-eBFP2 via sequencing AmpR/eBFP2 region<br>
| |
| - | <br>
| |
| - | 6.26<br>
| |
| - | AD blood cells from PrecisionMed? Talk to Brian?<br>
| |
| - | Meet with Kyle about designing siRNA (email sent)<br>
| |
| - | Make siRNA sequences in geneious<br>
| |
| - | Send order to Brian<br>
| |
| - | Picture of how low sensors come together<br>
| |
| - | Determine which pairs of target sites we want<br>
| |
| - | Find sequences (hopefully under 200 bp)<br>
| |
| - | Send ultramer order to Brian<br>
| |
| - | Plan new MAV vector<br>
| |
| - | Plan high sensor construction (meet with Brian)<br>
| |
| - | Golden Gate<br>
| |
| - | Gather miRNA target sites from Jeremy<br>
| |
| - | Nanodrop for concentrations ( ? )<br>
| |
| - | Make all 6 single low sensors (in conjunction with antibody group)<br>
| |
| - | Miniprep eBFP2<br>
| |
| - | NOTES<br>
| |
| - | Both LR's failed (mKate2 and L7ae)<br>
| |
| - | Meeting w/ Jeremy tomorrow to talk about new MAV vector and high sensors<br>
| |
| - | We need to Gibson in eBFP2 before we can finish the sensors<br>
| |
| - | <br>
| |
| - | siRNA MEETING WITH KYLE<br>
| |
| - | mature (5' phospho) and sense (reverse complement)<br>
| |
| - | copy format from 2013<br>
| |
| - | send brian both sequences as a duplex (IDT has a duplex option), with all the modification<br>
| |
| - | We NEED to Gibson in the eBFP2 control<br>
| |
| - | each high sensor on its own transcript (not necessarily own plasmid)<br>
| |
| - | QUESTIONS<br>
| |
| - | what do the "r" and "m" stand for?<br>
| |
| - | -rna and modification<br>
| |
| - | where do the overhangs come from? why are they there?<br>
| |
| - | -mature one has 2 u's (constant)<br>
| |
| - | 6.27<br>
| |
| - | Write up low sensor experiment plans<br>
| |
| - | Keep updating the parts list<br>
| |
| - | Meet with Jeremy<br>
| |
| - | Discuss high sensor plans<br>
| |
| - | New MAV assembly plan<br>
| |
| - | Questions for Brian<br>
| |
| - | eBFP2 Gibson for Flow Cytometry ( ? )<br>
| |
| - | Send siRNA sequences<br>
| |
| - | Miniprep Hef1a<br>
| |
| - | Golden Gate all the low sensors<br>
| |
| - | NOTES<br>
| |
| - | Reasons to not use phages for circuit delivery<br>
| |
| - | 6.30<br>
| |
| - | Keep updating the parts list<br>
| |
| - | Strengthen experiments page<br>
| |
| - | Prepare protocol for building new MAV<br>
| |
| - | Prepare protocol for building high sensors<br>
| |
| - | Transform low sensor Golden Gates<br>
| |
| - | Start building new MAV<br>
| |
| - | <br>
| |
| - | <br>
| |
| - | NOTES<br>
| |
| - | Meet w/ Brian tomorrow to talk about double input ultramer<br>
| |
| - | Co-transfect 2x Kturn & MAV w/ target sites & eBFP2<br>
| |
| - | PCR for building new MAV <br>
| |
| - | <br>
| |
| - | 7.1<br>
| |
| - | Keep updating the parts list<br>
| |
| - | Meet w/ Brian about double input low sensor<br>
| |
| - | Fill out PCR protocol to be ready for tomorrow<br>
| |
| - | Create high sensor construction map (unified to look like the other groups)<br>
| |
| - | Design primers / find restriction enzymes to sequence low sensors<br>
| |
| - | Pick multiple low sensor colonies<br>
| |
| - | Re-transform low sensors<br>
| |
| - | NOTES<br>
| |
| - | Double input low sensor will be a gBlock<br>
| |
| - | Lots of blue colonies, not many white colonies (none from GH)<br>
| |
| - | <br>
| |
| - | 7.2<br>
| |
| - | Keep updating the parts tree<br>
| |
| - | Propose experiments to test L7ae and Tau14/21<br>
| |
| - | Find restriction enzymes to digest original EF<br>
| |
| - | Miniprep original EF<br>
| |
| - | PCR mRFP out of pL1F_1_S6_S7<br>
| |
| - | PCR Purify mRFP<br>
| |
| - | Run the gel for mRFP<br>
| |
| - | Re Golden Gate EF and GH<br>
| |
| - | Digest mRFP cassette<br>
| |
| - | Re ligate mRFP cassette<br>
| |
| - | Transform mRFP cassette<br>
| |
| - | Retransform EF and GH<br>
| |
| - | NOTES<br>
| |
| - | There were no colonies to pick from EF and GH --> re transform<br>
| |
| - | <br>
| |
| - | 7.7<br>
| |
| - | Keep updating the parts tree (include experiments)<br>
| |
| - | Update experiments page<br>
| |
| - | Obtain plasmid maps<br>
| |
| - | Email Jin about TAL14 and miRNA target sites<br>
| |
| - | Talk to Lyla/Alexa about transfecting later this week<br>
| |
| - | Look for plasmids<br>
| |
| - | pEXPR: UAS-Gal4-TAL14-eYFP<br>
| |
| - | pEXPR: Gal4VP16<br>
| |
| - | pEXPR: Hef1a-TAL14<br>
| |
| - | pEXPR: Hef1a-2x Kturn-eYFP<br>
| |
| - | Transformations<br>
| |
| - | Low Sensor Golden Gates<br>
| |
| - | pEXPR: Hef1a-eBFP2 (186 ng/uL)<br>
| |
| - | pEXPR: Hef1a-L7ae (96 ng/uL)<br>
| |
| - | Pick red colonies and grow in medium!<br>
| |
| - | Find concentrations of eBFP2 and L7ae<br>
| |
| - | NOTES<br>
| |
| - | Nelson doesn't know where it is, but he needs it as well, so he's on the hunt<br>
| |
| - | If we're ready, we can transfect very early next week<br>
| |
| - | <br>
| |
| - | 7.8<br>
| |
| - | Keep updating the parts tree (include experiments)<br>
| |
| - | Figure out where the Golden Gates went<br>
| |
| - | Create protocol for siRNA transfection<br>
| |
| - | Meet with Jin at 3<br>
| |
| - | Transformations <br>
| |
| - | EF<br>
| |
| - | GH<br>
| |
| - | Mutant Hef1a-2xKturn-eGFP<br>
| |
| - | Wild Type Hef1a-2xKturn-eGFP<br>
| |
| - | Pick colonies from L7ae and eBFP2<br>
| |
| - | Miniprep MAV1212<br>
| |
| - | Digest MAV1212<br>
| |
| - | Run gel for MAV1212<br>
| |
| - | Image gel for MAV1212<br>
| |
| - | NOTES<br>
| |
| - | Jin gave us ideas to optimize our high sensors and experiments<br>
| |
| - | <br>
| |
| - | 7.9<br>
| |
| - | Keep updating the parts tree (include experiments)<br>
| |
| - | Find new miRNA target site vector. Email:<br>
| |
| - | Kyle<br>
| |
| - | Lila<br>
| |
| - | Samira<br>
| |
| - | Obtain plasmid maps<br>
| |
| - | Hef1a-2xKturn (mutant and w.t.)<br>
| |
| - | GAL4 (from Brian)<br>
| |
| - | Pick colonies & Grow in medium<br>
| |
| - | EF<br>
| |
| - | GH<br>
| |
| - | pENTR: L7ae<br>
| |
| - | pEXPR: Hef1a-eBFP2<br>
| |
| - | Mutant Hef1a-2xKturn-eGFP<br>
| |
| - | Wild Type Hef1a-2xKturn-eGFP<br>
| |
| - | MAV12112-Hef1a-eBFP2 (control)<br>
| |
| - | Create pENTR: TAL14<br>
| |
| - | BP Reaction from TAL14<br>
| |
| - | Transform TAL14<br>
| |
| - | NOTES<br>
| |
| - | GH and pENTR: L7ae didn't grow properly (their plates had almost no agar)<br>
| |
| - | L7ae was re transformed<br>
| |
| - | <br>
| |
| - | 7.10<br>
| |
| - | Keep updating the parts tree (include experiments)<br>
| |
| - | More descriptive names<br>
| |
| - | Include other plasmid<br>
| |
| - | Alternative backbones? Check iGEM 2013<br>
| |
| - | Plan experiment to test Kturn (Just Kturn and L7ae)<br>
| |
| - | Re-make double input low sensor as two ultramers (use the Q3 site)<br>
| |
| - | Pick colonies & grow in medium (pick 5)<br>
| |
| - | Midi prep<br>
| |
| - | EF<br>
| |
| - | GH<br>
| |
| - | pEXPR: Hef1a-eBFP2<br>
| |
| - | Mutant Hef1a-2xKturn-eGFP<br>
| |
| - | Wild Type Hef1a-2xKturn-eGFP<br>
| |
| - | Mini prep<br>
| |
| - | pENTR: L7ae<br>
| |
| - | GH<br>
| |
| - | Mini prep<br>
| |
| - | EF<br>
| |
| - | pEXPR: Hef1a-eBFP2<br>
| |
| - | Mutant Hef1a-2xKturn-eGFP<br>
| |
| - | Wild Type Hef1a-2xKturn-eGFP<br>
| |
| - | LR<br>
| |
| - | MAV1212-Hef1a-L7ae<br>
| |
| - | MAV1212-Hef1a-TAL14<br>
| |
| - | BP<br>
| |
| - | P4-donor-P1R on Brian's bench<br>
| |
| - | TAL14-mKate pEST in Brian's fridge<br>
| |
| - | Golden Gate<br>
| |
| - | GH<br>
| |
| - | Transformation<br>
| |
| - | TAL14 Promoter (from BP rxn)<br>
| |
| - | TAL14-mKate-pest<br>
| |
| - | pENTR: L1_TAL14_L2<br>
| |
| - | MAV1212-mRFP<br>
| |
| - | NOTES<br>
| |
| - | We should sequence the low sensors when they're done<br>
| |
| - | Golden Gate Calculations<br>
| |
| - | <br>
| |
| - | 7.11<br>
| |
| - | Keep updating the parts tree (include experiments)<br>
| |
| - | Alternative backbones? Check iGEM 2013<br>
| |
| - | Actual list of experiments<br>
| |
| - | TAL14 & GAL4 (to test promoter)<br>
| |
| - | MAV1212-hEF1a-eBFP2<br>
| |
| - | Make/Find primers<br>
| |
| - | To sequence both low sensors<br>
| |
| - | Sequence/digest plasmids to verify - may be unnecessary. Emailed Brian.<br>
| |
| - | EF<br>
| |
| - | GH - don't have this<br>
| |
| - | pEXPR: MAV1212-Hef11a-eBFP2<br>
| |
| - | Mutant Hef1a-2xKturn-eGFP<br>
| |
| - | Wild Type Hef1a-2xKturn-eGFP<br>
| |
| - | Pick colonies and grow up<br>
| |
| - | MAV1212-mRFP<br>
| |
| - | L1_TALER14_L2<br>
| |
| - | pTAL14-mKate-pEST<br>
| |
| - | MAV1212-Hef1a-eBFP2 for midiprep (pending Brian's approval)<br>
| |
| - | EF low sensor for midiprep (pending Brian's approval)<br>
| |
| - | Mutant Hef1a-2xKturn-eGFP for midiprep (pending Brian's approval)<br>
| |
| - | WT Hef1a-2xKturn-eGFP for midiprep (pending Brian's approval)<br>
| |
| - | Miniprep<br>
| |
| - | GH - don't have this<br>
| |
| - | Transformations<br>
| |
| - | GH (Golden Gate)<br>
| |
| - | Hef1a-L7ae (LR)<br>
| |
| - | Hef1a-TAL14 (LR)<br>
| |
| - | BP<br>
| |
| - | P4-donor-P1R with TAL14-mKate pEST<br>
| |
| - | NOTES<br>
| |
| - | BP rxn didn't yield colonies --> re-doing it and letting it grow overnight this time<br>
| |
| - | GH plate has no colonies :/<br>
| |
| - | <br>
| |
| - | 7.14<br>
| |
| - | Keep updating the parts tree (include experiments)<br>
| |
| - | Obtain pEXPR: hEF1a-GAL4VP16<br>
| |
| - | Fill out the Genewiz Order Form to sequence Low Sensor miR-144<br>
| |
| - | Prepare transfection well plate maps<br>
| |
| - | So where are the siRNA though...<br>
| |
| - | Why didn't the GH golden gate work on try #3?<br>
| |
| - | Change name of high sensors in tree<br>
| |
| - | Pick colonies<br>
| |
| - | pEXPR: TAL14-mKate<br>
| |
| - | Midiprep<br>
| |
| - | pEXPR: MAV1212-Hef11a-eBFP2<br>
| |
| - | Mutant Hef1a-2xKturn-eGFP<br>
| |
| - | Wild Type Hef1a-2xKturn-eGFP<br>
| |
| - | Low Sensor miR-144<br>
| |
| - | Hef1a-L7ae (LR)<br>
| |
| - | Miniprep<br>
| |
| - | MAV1212-mRFP<br>
| |
| - | L1_TALER14_L2<br>
| |
| - | pTAL14-mKate-pEST<br>
| |
| - | LR<br>
| |
| - | pEXPR: MAV1212-TRE-L7ae<br>
| |
| - | NOTES<br>
| |
| - | so...the siRNA are nowhere to be found<br>
| |
| - | hEF1a-TAL14 (LR) has no colonies<br>
| |
| - | TRE is nowhere to be found<br>
| |
| - | <br>
| |
| - | 7.15<br>
| |
| - | Keep updating the parts tree (include experiments)<br>
| |
| - | Obtain Plasmids<br>
| |
| - | pEXPR: hEF1a-GAL4VP16<br>
| |
| - | pENTR: TRE (promoter)<br>
| |
| - | pEXPR: TAL14-mKate<br>
| |
| - | Troubleshoot<br>
| |
| - | 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>
| |
| - | Retransform products at higher concentration<br>
| |
| - | Fill out the Genewiz Order Form to sequence Low Sensor miR-144<br>
| |
| - | Design primers to sequence high sensors<br>
| |
| - | Ask Jeremy about sequencing primers<br>
| |
| - | Look into siRNA transfection<br>
| |
| - | TOP PRIORITY: SEQUENCE LOW SENSORS (can't be done till primers arrive)<br>
| |
| - | Pick colonies<br>
| |
| - | pEXPR: TAL14-mKate<br>
| |
| - | LR<br>
| |
| - | pEXPR: MAV1212-TRE-L7ae<br>
| |
| - | Transfection<br>
| |
| - | L7ae experiment<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>
| |
| - | Combined Low Sensor miR-144-miR-181c<br>
| |
| - | Get ultramers from Brian<br>
| |
| - | Anneal target sites<br>
| |
| - | All high sensors<br>
| |
| - | Transformation<br>
| |
| - | Re-do Low Sensor miR-181c<br>
| |
| - | Midiprep<br>
| |
| - | pEXPR: TAL14-mKate<br>
| |
| - | NOTES<br>
| |
| - | The transfections are a little bit messed up<br>
| |
| - | We need to verify the MAV1212-hEF1a-L7ae<br>
| |
| - | <br>
| |
| - | 7.16<br>
| |
| - | 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>
| |
| - | 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>
| |
| - | <br>
| |
| - | <br>June 15
| |
| - | <br>AG, LA SS
| |
| - | <br>Grew pENTR_LilrB2, pENTR_PirB
| |
| - | <br>
| |
| - | <br>June 16
| |
| - | <br>Liquid cultures didn't grow
| |
| - | <br>SS
| |
| - | <br>New liquid cultures made
| |
| - | <br>AG, LA
| |
| - | <br>NEGEM and High School presentations completed
| |
| - | <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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