Team:MIT/Notebook

From 2014.igem.org

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<h2>miRNA</h2>
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<table width=100% style="background-color:#c5c5c5"><tr>
+
6.22<br>
-
<td class="links" >
+
Update the parts list on the wiki<br>
-
<ul>
+
Create an experiments page on the wiki<br>
-
<li><a href="#top">Top</a></li>
+
Prepare to meet with Jake Beal<br>
-
<li><a href="#description">Description</a></li>
+
Re-transform MAV1212<br>
-
<li><a href="#outcome">Outcome</a></li>
+
Re-transform MAV1212-Hef1a-eBFP2<br>
-
<li><a href="#experiments">Experiments</a>
+
Mini prep MAV1212-Hef1a-eBFP2<br>
-
<ul>
+
6.23<br>
-
<li><a href="#1">Localization</a></li>
+
Go over workflow of plasmid construction as a group<br>
-
<li><a href="#2">Binding</a></li>
+
Finish a unified daily log<br>
-
<li><a href="#3">Cofilin levels</a></li>
+
Digest meeting with Jake Beal<br>
-
<li><a href="#4">Baseline</a></li>
+
Go through plates<br>
-
<li><a href="#5">Activation</a></li>
+
Re plate MAV1212<br>
-
</ul>
+
Pick colonies for MAV1212<br>
-
</li>
+
Plan LacZ PCR product<br>
-
<li><a href="#parts">Parts</a></li>
+
Primers ( ? )<br>
-
</ul>
+
Figure out what's going on<br>
-
</td>
+
<br>
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<td width="865px" style="background-color:#f3f3f3;padding-left:25px;padding-right:25px;">
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6.24<br>
-
 
+
Present about cell models<br>
-
</td></tr>
+
COMPLETE PARTS LIST ON WIKI<br>
-
</table>
+
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)
</body>
</body>
</html>
</html>

Revision as of 02:17, 17 October 2014

 


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miRNA

6.22
Update the parts list on the wiki
Create an experiments page on the wiki
Prepare to meet with Jake Beal
Re-transform MAV1212
Re-transform MAV1212-Hef1a-eBFP2
Mini prep MAV1212-Hef1a-eBFP2
6.23
Go over workflow of plasmid construction as a group
Finish a unified daily log
Digest meeting with Jake Beal
Go through plates
Re plate MAV1212
Pick colonies for MAV1212
Plan LacZ PCR product
Primers ( ? )
Figure out what's going on

6.24
Present about cell models
COMPLETE PARTS LIST ON WIKI
Prep experiments
Contact Jeremy about multiple miRNA target sites chained together
Pick colonies for MAV1212
Miniprep MAV1212
Keep MAV1212-Hef1a-eBFP2 (for use WITHOUT target sites)
Gateway MAV1212-Hef1a-L7ae (for high sensor)
Gateway MAV1212_Hef1a-eYFP (for use WITH target sites)
Transform Hef1a, MAV1212 ( ? )
NOTES:
No MAV1212 colonies grew on the plates or in the medium
Jeremy gave us more, enough for one more transformation
6.25
Create a clear workflow
Update "status" of plasmids on parts list
How do we string multiple low sensors together?
Determine which pairs of target sites we want
Find sequences (hopefully under 200 bp)
Send ultramer order to Brian
Tranform LRs
MAV1212-Hef1a-mKate2
MAV1212-Hef1a-L7ae
Hef1a on Kan
Pick colonies from transformations
MAV1212-Hef1a-eBFP2
Golden Gate
Gather miRNA target sites from Jeremy
Make all 6 single low sensors
NOTES
MAV1212 didn't grow (ccdB is over expressed)
Hef1a was plated on Amp plates...but it has Kan resistance
Verify MAV1212-Hef1a-eBFP2 via sequencing AmpR/eBFP2 region

6.26
AD blood cells from PrecisionMed? Talk to Brian?
Meet with Kyle about designing siRNA (email sent)
Make siRNA sequences in geneious
Send order to Brian
Picture of how low sensors come together
Determine which pairs of target sites we want
Find sequences (hopefully under 200 bp)
Send ultramer order to Brian
Plan new MAV vector
Plan high sensor construction (meet with Brian)
Golden Gate
Gather miRNA target sites from Jeremy
Nanodrop for concentrations ( ? )
Make all 6 single low sensors (in conjunction with antibody group)
Miniprep eBFP2
NOTES
Both LR's failed (mKate2 and L7ae)
Meeting w/ Jeremy tomorrow to talk about new MAV vector and high sensors
We need to Gibson in eBFP2 before we can finish the sensors

siRNA MEETING WITH KYLE
mature (5' phospho) and sense (reverse complement)
copy format from 2013
send brian both sequences as a duplex (IDT has a duplex option), with all the modification
We NEED to Gibson in the eBFP2 control
each high sensor on its own transcript (not necessarily own plasmid)
QUESTIONS
what do the "r" and "m" stand for?
-rna and modification
where do the overhangs come from? why are they there?
-mature one has 2 u's (constant)
6.27
Write up low sensor experiment plans
Keep updating the parts list
Meet with Jeremy
Discuss high sensor plans
New MAV assembly plan
Questions for Brian
eBFP2 Gibson for Flow Cytometry ( ? )
Send siRNA sequences
Miniprep Hef1a
Golden Gate all the low sensors
NOTES
Reasons to not use phages for circuit delivery
6.30
Keep updating the parts list
Strengthen experiments page
Prepare protocol for building new MAV
Prepare protocol for building high sensors
Transform low sensor Golden Gates
Start building new MAV


NOTES
Meet w/ Brian tomorrow to talk about double input ultramer
Co-transfect 2x Kturn & MAV w/ target sites & eBFP2
PCR for building new MAV

7.1
Keep updating the parts list
Meet w/ Brian about double input low sensor
Fill out PCR protocol to be ready for tomorrow
Create high sensor construction map (unified to look like the other groups)
Design primers / find restriction enzymes to sequence low sensors
Pick multiple low sensor colonies
Re-transform low sensors
NOTES
Double input low sensor will be a gBlock
Lots of blue colonies, not many white colonies (none from GH)

7.2
Keep updating the parts tree
Propose experiments to test L7ae and Tau14/21
Find restriction enzymes to digest original EF
Miniprep original EF
PCR mRFP out of pL1F_1_S6_S7
PCR Purify mRFP
Run the gel for mRFP
Re Golden Gate EF and GH
Digest mRFP cassette
Re ligate mRFP cassette
Transform mRFP cassette
Retransform EF and GH
NOTES
There were no colonies to pick from EF and GH --> re transform

7.7
Keep updating the parts tree (include experiments)
Update experiments page
Obtain plasmid maps
Email Jin about TAL14 and miRNA target sites
Talk to Lyla/Alexa about transfecting later this week
Look for plasmids
pEXPR: UAS-Gal4-TAL14-eYFP
pEXPR: Gal4VP16
pEXPR: Hef1a-TAL14
pEXPR: Hef1a-2x Kturn-eYFP
Transformations
Low Sensor Golden Gates
pEXPR: Hef1a-eBFP2 (186 ng/uL)
pEXPR: Hef1a-L7ae (96 ng/uL)
Pick red colonies and grow in medium!
Find concentrations of eBFP2 and L7ae
NOTES
Nelson doesn't know where it is, but he needs it as well, so he's on the hunt
If we're ready, we can transfect very early next week

7.8
Keep updating the parts tree (include experiments)
Figure out where the Golden Gates went
Create protocol for siRNA transfection
Meet with Jin at 3
Transformations
EF
GH
Mutant Hef1a-2xKturn-eGFP
Wild Type Hef1a-2xKturn-eGFP
Pick colonies from L7ae and eBFP2
Miniprep MAV1212
Digest MAV1212
Run gel for MAV1212
Image gel for MAV1212
NOTES
Jin gave us ideas to optimize our high sensors and experiments

7.9
Keep updating the parts tree (include experiments)
Find new miRNA target site vector. Email:
Kyle
Lila
Samira
Obtain plasmid maps
Hef1a-2xKturn (mutant and w.t.)
GAL4 (from Brian)
Pick colonies & Grow in medium
EF
GH
pENTR: L7ae
pEXPR: Hef1a-eBFP2
Mutant Hef1a-2xKturn-eGFP
Wild Type Hef1a-2xKturn-eGFP
MAV12112-Hef1a-eBFP2 (control)
Create pENTR: TAL14
BP Reaction from TAL14
Transform TAL14
NOTES
GH and pENTR: L7ae didn't grow properly (their plates had almost no agar)
L7ae was re transformed

7.10
Keep updating the parts tree (include experiments)
More descriptive names
Include other plasmid
Alternative backbones? Check iGEM 2013
Plan experiment to test Kturn (Just Kturn and L7ae)
Re-make double input low sensor as two ultramers (use the Q3 site)
Pick colonies & grow in medium (pick 5)
Midi prep
EF
GH
pEXPR: Hef1a-eBFP2
Mutant Hef1a-2xKturn-eGFP
Wild Type Hef1a-2xKturn-eGFP
Mini prep
pENTR: L7ae
GH
Mini prep
EF
pEXPR: Hef1a-eBFP2
Mutant Hef1a-2xKturn-eGFP
Wild Type Hef1a-2xKturn-eGFP
LR
MAV1212-Hef1a-L7ae
MAV1212-Hef1a-TAL14
BP
P4-donor-P1R on Brian's bench
TAL14-mKate pEST in Brian's fridge
Golden Gate
GH
Transformation
TAL14 Promoter (from BP rxn)
TAL14-mKate-pest
pENTR: L1_TAL14_L2
MAV1212-mRFP
NOTES
We should sequence the low sensors when they're done
Golden Gate Calculations

7.11
Keep updating the parts tree (include experiments)
Alternative backbones? Check iGEM 2013
Actual list of experiments
TAL14 & GAL4 (to test promoter)
MAV1212-hEF1a-eBFP2
Make/Find primers
To sequence both low sensors
Sequence/digest plasmids to verify - may be unnecessary. Emailed Brian.
EF
GH - don't have this
pEXPR: MAV1212-Hef11a-eBFP2
Mutant Hef1a-2xKturn-eGFP
Wild Type Hef1a-2xKturn-eGFP
Pick colonies and grow up
MAV1212-mRFP
L1_TALER14_L2
pTAL14-mKate-pEST
MAV1212-Hef1a-eBFP2 for midiprep (pending Brian's approval)
EF low sensor for midiprep (pending Brian's approval)
Mutant Hef1a-2xKturn-eGFP for midiprep (pending Brian's approval)
WT Hef1a-2xKturn-eGFP for midiprep (pending Brian's approval)
Miniprep
GH - don't have this
Transformations
GH (Golden Gate)
Hef1a-L7ae (LR)
Hef1a-TAL14 (LR)
BP
P4-donor-P1R with TAL14-mKate pEST
NOTES
BP rxn didn't yield colonies --> re-doing it and letting it grow overnight this time
GH plate has no colonies :/

7.14
Keep updating the parts tree (include experiments)
Obtain pEXPR: hEF1a-GAL4VP16
Fill out the Genewiz Order Form to sequence Low Sensor miR-144
Prepare transfection well plate maps
So where are the siRNA though...
Why didn't the GH golden gate work on try #3?
Change name of high sensors in tree
Pick colonies
pEXPR: TAL14-mKate
Midiprep
pEXPR: MAV1212-Hef11a-eBFP2
Mutant Hef1a-2xKturn-eGFP
Wild Type Hef1a-2xKturn-eGFP
Low Sensor miR-144
Hef1a-L7ae (LR)
Miniprep
MAV1212-mRFP
L1_TALER14_L2
pTAL14-mKate-pEST
LR
pEXPR: MAV1212-TRE-L7ae
NOTES
so...the siRNA are nowhere to be found
hEF1a-TAL14 (LR) has no colonies
TRE is nowhere to be found

7.15
Keep updating the parts tree (include experiments)
Obtain Plasmids
pEXPR: hEF1a-GAL4VP16
pENTR: TRE (promoter)
pEXPR: TAL14-mKate
Troubleshoot
So where are the siRNA though... (keep looking around, they were ordered)
Why didn't the GH golden gate work on try #3?
Retransform products at higher concentration
Fill out the Genewiz Order Form to sequence Low Sensor miR-144
Design primers to sequence high sensors
Ask Jeremy about sequencing primers
Look into siRNA transfection
TOP PRIORITY: SEQUENCE LOW SENSORS (can't be done till primers arrive)
Pick colonies
pEXPR: TAL14-mKate
LR
pEXPR: MAV1212-TRE-L7ae
Transfection
L7ae experiment
Golden Gate
High Sensor miR-30d (I13 & J13)
High Sensor miR-146a (I5 & J5)
High Sensor let-7f (1.13 & 1.14)
High Sensor miR-125b (A3 & B3)
Combined Low Sensor miR-144-miR-181c
Get ultramers from Brian
Anneal target sites
All high sensors
Transformation
Re-do Low Sensor miR-181c
Midiprep
pEXPR: TAL14-mKate
NOTES
The transfections are a little bit messed up
We need to verify the MAV1212-hEF1a-L7ae

7.16
How To Please Ron/Brain
Keep updating the parts tree (include experiments)
miRNA Sensors (Theory)
Anaylze Low Sensor miR-144 sequencing results
miRNA Sensors (Lab Work)
Restriction Digest
MAV1212-hEF1a-L7ae / that other shit in the fridge
Golden Gate
High Sensor miR-30d (I13 & J13)
High Sensor miR-146a (I5 & J5)
High Sensor let-7f (1.13 & 1.14)
High Sensor miR-125b (A3 & B3)
Low Sensor miR-144 (E5 & F5)
Low Sensor miR-181c (G7 & H7)
Low Sensor miR-144-miR-181c
Transform
pEXPR: MAV1212-TRE-L7ae (LR)
pEXPR: MAV1212-hEF1a-GAL4VP16 (LR)
pEXPR: MAV1212-TRE-GAL4VP16 (LR)
Re-suspend Low Sensor miR-144-181c oligos
Anneal Low Sensor miR-44-181c oligos
NOTES
Low Sensor miR-144 did not pass quality control for sequencing
After annealing the double input low sensors, the didn't nanodrop well
Golden Gate Calculations
Restriction Digest Calculations

7.17
�
Keep updating the parts tree (include experiments)
Troubleshoot
Why didn't the oligos nanodrop??
miRNA Sensors (Lab Work)
Golden Gate
Low Sensor miR-144-miR-181c
Transform
High Sensor miR-30d
High Sensor miR-146a
High Sensor let-7f
High Sensor miR-125b
Low Sensor miR-144
Low Sensor miR-181c
Pick colonies to miniprep and midiprep
pEXPR: MAV1212-TRE-L7ae (LR)
pEXPR: MAV1212-hEF1a-GAL4VP16 (LR)
pEXPR: MAV1212-TRE-GAL4VP16 (LR)
Prepare and run a gel (Hyperladder 2 and 4% agaros gel w/ annealed target sites)
High Sensor miR-30d TS
High Sensor miR-146a TS
High Sensor let-7f TS
High Sensor miR-125b TS
Low Sensor miR-144 TS
Low Sensor miR-181c TS
FACS cell prep
Transfect L7ae Experiment
Analyze FACS data
NOTES
We have a new primer to sequence with (again, from Jeremy)
We now have FACS DATA
7.18
Keep updating the parts tree (include experiments)
Troubleshoot
Why didn't the oligos nanodrop??
Meet w/ Kyle about FlowJo
Look for the siRNA
Golden Gate
Low Sensor miR-144-miR-181c
Pick colonies
High Sensor miR-30d
High Sensor miR-146a
High Sensor let-7f
High Sensor miR-125b
Low Sensor miR-144
Low Sensor miR-181c
Miniprep
pEXPR: MAV1212-TRE-L7ae (LR)
pEXPR: MAV1212-hEF1a-GAL4VP16 (LR)
pEXPR: MAV1212-TRE-GAL4VP16 (LR)
Restriction Digest & run gel
pEXPR: MAV1212-TRE-L7ae (LR)
pEXPR: MAV1212-hEF1a-GAL4VP16 (LR)
pEXPR: MAV1212-TRE-GAL4VP16 (LR)
Midiprep?
pEXPR: MAV1212-TRE-L7ae (LR)
pEXPR: MAV1212-hEF1a-GAL4VP16 (LR)
pEXPR: MAV1212-TRE-GAL4VP16 (LR)
Gateway
pEXPR: MAV1212-hEF1a-TAL14
Transform
pENTR: TAL14
pENTR: TAL21
pENTR: Something for yeast for Brian
NOTES
The gel didn't work very well, so we're re-doing it tomorrow
7.20
Keep updating the parts tree (include experiments)
Meet w/ Kyle about FlowJo
Troubleshoot
What's going on with the double input low sensor
Move siRNA into our cryobox
Pick colonies
High Sensor miR-30d
High Sensor miR-146a
High Sensor let-7f
High Sensor miR-125b
Low Sensor miR-144
Low Sensor miR-181c
pENTR: TAL14
pENTR: TAL21
pENTR: Something for yeast for Brian
Restriction Digest & run gel
pEXPR: MAV1212-TRE-L7ae (LR)
pEXPR: MAV1212-hEF1a-GAL4VP16 (LR)
pEXPR: MAV1212-TRE-GAL4VP16 (LR)
Inoculate midiprep culture?
If the LR's have the right sequence
Transform
Re transform MAV1212-hEF1a-TAL14 LR
FACS Prep
NOTES
The digest keeps failing

7.21
Keep updating the parts tree (include experiments)
Meet w/ Kyle
FlowJo
Bright Field Images
miRNA Sensors (Theory)
Troubleshoot
What's going on with the double input low sensor
Is there a different GG thermal cycler program for Bbs1?
Run new transfection plate/protocol by Brian
Move siRNA into our cryobox
Grab sequencing primer from Jeremy's bench
Pick colonies
pEXPR: MAV1212-hEF1a-TAL14 (Mini)
Give plates to Brian
pENTR: TAL14p
pENTR: TAL21p
pENTR: Something for yeast for Brian
Restriction Digest & run gel
pEXPR: MAV1212-TRE-L7ae (LR)
pEXPR: MAV1212-TRE-GAL4VP16 (LR)
Transform
pEXPR: MAV1212-hEF1a-GAL4VP16
Golden Gate
All the sensors
Split and Seed 4 plates
NOTES
We transfected way too much DNA --> new protocol with 1 ug total

7.22
Keep updating the parts tree (include experiments)
Meet w/ Kyle
FlowJo
Bright Field Images
miRNA Sensors (Theory)
Troubleshoot
What's going on with the double input low sensor
Run new transfection plate/protocol by Brian
Move siRNA into our cryobox
Grab sequencing primer from Jeremy's bench
Miniprep
pEXPR: MAV1212-hEF1a-TAL14
Restriction Digest & run gel (4 samples)
pEXPR: MAV1212-TRE-L7ae (LR)
Transform
pEXPR: MAV1212-TRE-GAL4VP16 (LR)
All the low sensors
Transfect for L7ae Experiment 1.3


NOTES
We ran out of Lipofectamine and couldn't transfect

7.24
How To Please Ron/Brain
Keep updating the parts tree (include experiments)
miRNA Sensors (Theory)
miRNA Sensors (Lab Work)
Midi prep
pEXPR: MAV1212-hEF1a-TAL14
Transform
pEXPR: MAV1212-TRE-L7ae (LR from last night)
Proteinase K
MAV1212-hEF1a-TAL14
Restriction Digest and run gel
MAV1212-hEF1a-TAL14
LR
MAV1212-hEF1a-TAL21
Mini prep
Low Sensor miR-144
Low Sensor miR-181c
High Sensor miR-30d
High Sensor miR-125b
High Sensor miR-146a
High Sensor let-7f
Send for sequencing
Low Sensor miR-144
Low Sensor miR-181c
High Sensor miR-30d
High Sensor miR-125b
High Sensor miR-146a
High Sensor let-7f
Pick colonies
Low Sensor miR-144
Low Sensor miR-181c
High Sensor miR-30d
High Sensor miR-125b
High Sensor miR-146a
High Sensor let-7f
pEXPR: MAV1212-hEF1a-GAL4VP16
pEXPR: MAV1212-TRE-GAL4VP16
Seed plates
L7ae experiment (Take 3)
NOTES
pEXPR: MAV1212-hEF1a-GAL4VP16 and pEXPR: MAV1212-TRE-GAL4VP16 only had blue colonies, which doesn't make sense because they were both verified...
Brian is in the middle of something on his bench, so we won't midi today

7.25
Keep updating the parts tree (include experiments)
Troubleshoot
What's the deal with the GAL4VP16?!
Pick colonies
pEXPR: MAV1212-TRE-L7ae (mini)
MAV1212-hEF1a-GAL4VP16 (midi)
MAV1212-TRE-GAL4VP16 (midi)
Proteinase K
MAV1212-hEF1a-TAL21
MAV1212-hEF1a-TAL14
Transform
MAV1212-hEF1a-TAL21 (LR)
MAV1212-hEF1a-TAL14 (LR)
Plasmids from BCR group
Plasmids from protein receptor group
Plasmids from Brian
Miniprep
All the sensors (round 2)
Send for sequencing
All the sensors (round 2)
Analyze sequencing results
All the sensors (round 1)
Transfect
L7ae Experiment (Take 3)
Seed
TAL14 Experiment (Take 1)
Re-anneal
miR-144-181c double input target site
Golden Gate
Low Sensor miR-144-181c
Inoculate midi cultures
Each of the single input sensors
NOTES
All of the single input sensors were verified!
1st full day with no hiccups?
Gave constructs and plasmids maps (Kturns, L7ae, and MAV RFP) to Jeremy
7.26
Keep updating the parts tree (include experiments)
Mini prep
pEXPR: MAV1212-TRE-L7ae
Digest and run gel
pEXPR: MAV1212-TRE-L7ae
Inoculate midi (if digest is okay)
pEXPR: MAV1212-TRE-L7ae
Midi prep
MAV1212-hEF1a-GAL4VP16
MAV1212-TRE-GAL4VP16
All the single input sensors
Pick colonies
MAV1212-hEF1a-TAL21
MAV1212-hEF1a-TAL14
Plasmids from BCR group
Plasmids from protein receptor group
Plasmids from Brian
Analyze sequencing results
All the sensors (round 2)
Transform
Low Sensor miR-144-181c
Transfect
TAL14 Experiment (Take 1)
Seed
Experiment #3 (Take 1)
Experiment #4 (Take 2)
NOTES

7.28
Keep updating the parts tree (include experiments)
Include TAL14 and TAL21 high sensors
Analyze sequencing results
Plan DOX induction experiment for L7ae repression
Plan to build TAL14/TAL21 high sensors
Do gating and analysis of Exp 1 Attempt 3
Mini prep
pEXPR: MAV1212-TRE-L7ae
Digest and run gel
pEXPR: MAV1212-TRE-L7ae
Inoculate midi (if digest is okay)
pEXPR: MAV1212-TRE-L7ae
Pick colonies
MAV1212-hEF1a-TAL21
MAV1212-hEF1a-TAL14
Low Sensor miR-144-181c
Golden Gate
All the target sites & TAL14-mKate with Bbs1
NOTES
we're starting construction of the TAL14/21 high sensors

7.29
How To Please Ron/Brain
Keep updating the parts tree (include experiments)
Get TAL21 promoter from Brian
Plan experiments
DOX induction for L7ae repression (look here)
Tuning TAL14 repression

Learn about gating and FACS analysis
Midi prep
pEXPR: MAV1212-TRE-L7ae
Mini prep
MAV1212-hEF1a-TAL21
MAV1212-hEF1a-TAL14
Low Sensor miR-144-181c
Sequence verify
Low Sensor miR-144-181c
Digest and run gel
MAV1212-hEF1a-TAL21
MAV1212-hEF1a-TAL14
Golden Gate (if digest works)
All the TAL21 high sensors
Transform
All the TAL14 high sensors
And a plasmid from BCR
NOTES
The minipreps failed? re do tomorrow

7.30
Keep updating the parts tree (include experiments)
Run tuning experiments by Brian
Get transformation code from Brian (hopefully)
Midi prep
pEXPR: TAL21-mKate
Mini prep
MAV1212-hEF1a-TAL21
MAV1212-hEF1a-TAL14
Low Sensor miR-144-181c
Sequence verify
Low Sensor miR-144-181c
Digest and run gel
MAV1212-hEF1a-TAL21
MAV1212-hEF1a-TAL14
Golden Gate (if digest works)
All the TAL21 high sensors
Pick colonies
miR-30d (TAL14)
miR-125b (TAL14)
let-7f (TAL14)
TAL14 mKate
Inoculate midi culture (in case the digest works)
TAL21
NOTES
The midi prep for TAL21-mKate didn't work --> pick a colony and try again tomorrow
High sensor miR-146a didn't grow colonies --> retransform + replate transformation

7.31
Keep updating the parts tree (include experiments)
What about the double input low sensor?
Midi prep
pEXPR: TAL21-mKate
Run gel
MAV1212-hEF1a-TAL21
MAV1212-hEF1a-TAL14
Golden Gate (if digest works)
All the TAL21 high sensors
Gateway (LR)
pEXPR: hEF1a-TAL21
Pick colonies
miR-30d (TAL14)
miR-125b (TAL14)
let-7f (TAL14)
pEXPR: hEF1a-TAL21
DOX Induction
L7ae tuning experiment
Transfect
TAL14 tuning experiment
NOTES
hEF1a-TAL21 had a red pellet during midi prep spin down --> re-LR & re pick colonies

8.1
Keep updating the parts tree (include experiments)
Double input low sensor??
Where are the siRNA?
Mini prep
miR-30d (TAL14)
miR-16a (TAL14)
let-7f (TAL14)
pEXPR: hEF1a-TAL21
Send for sequencing
miR-30d (TAL14)
miR-16a (TAL14)
let-7f (TAL14)
Digest and Run gel
MAV1212-hEF1a-TAL21
Flow Cytometry
L7ae tuning experiment
Transform
miR-125b (TAL14)
NOTES
minipreps took a really long time --> TAL21 wasn't digested
siRNA should be coming "soon"
since HS_125b didn't grow colonies --> re transformed

8.4
Keep updating the parts tree (include experiments)
miRNA Sensors (Theory)
Meet with Kyle about quantifying repression
Troubleshoot gel results
Digest and run gel
MAV1212-hEF1a-TAL14
MAV1212-hEF1a-TAL21
LR (if digest fails)
MAV1212-hEF1a-TAL21 - find pENTR TAL21
Pick colonies (if digest failes)
pENTR TAL14
double input low sensor

8.7
Keep updating the parts tree (include experiments)
Meet with Kyle about quantifying repression
Miniprep
pENTR TAL14
Double-Input Low Sensor
Find
pEXPR hEF1A:TAL14
pENTR TAL21
pEXPR hEF1A:TAL21
LR
MAV1212-hEF1a-TAL14
MAV1212-hEF1a-TAL21
Sequence
Double-input low sensor
Digest & run gel
pENTR: TAL14
Transfect
L7ae quantification experiment
NOTES

8.8
Keep updating the parts tree (include experiments)
Figure out MATLAB quantification
Make plate maps for siRNA transfection
Analyze Digest
pENTR TAL14
Analyze Sequencing Results
Double-Input Low Sensor
Add DOX
L7ae quantification experiment
LR
hEF1a-TAL14 (if digest works)
hEF1a-TAL21 (find entry vector / plate)
NOTES

8.11
Keep updating the parts tree (include experiments)
FIND pENTR: TAL21
TASBE tools with Brian
Anaylze sequencing results
Pick up sensors from Jeremy
Clean up experiments page
Transform
hEF1a-TAL14 (LR)
Sensors from Jeremy?
Inoculate midi culture
Double input low sensor
Tissue Culture
Seed plate for miRNA sensors w/o siRNA
Check dilutions in TC room
NOTES

8.12
Keep updating the parts tree (include experiments)
FIND pENTR: TAL21
TASBE tools with Brian
Pick up sensors from Jeremy
Tissue Culture
Seed plate for miRNA sensors w/ siRNA??
Check dilutions in TC room
NOTES

8.13
Keep updating the parts tree (include experiments)
Emal Jake Beal about TASBE tools
Pick up sensors from Jeremy
Tissue Culture
Transfect plate for miRNA sensors w/ siRNA
Miniprep
hEF1a-TAL14
(Restriction Digest and Run Gel)
hEF1a-TAL14
(Golden Gate)
TAL14 High Sensors
NOTES

8.14
Keep updating the parts tree (include experiments)
Emal Jake Beal about TASBE tools
Pick up sensors from Jeremy
And figure out what/when to do with them...
New map for siRNA experiments
Re-name siRNAs as "siRNA-[insert number here]"
Figure out scatterplot in MATLAB
Tissue Culture
Cytometry for miRNA sensors w/o siRNA
Golden Gate
TAL14 High Sensors
Grow in midi culture
Double input low sensor
GG Donor for TC
hEF1a-TAL21

8.15
Keep updating the parts tree (include experiments)
Meet with Kyle about scatterplots in MATLAB
Tissue Culture
Cytometry for miRNA sensors with siRNA
Digest verify
MAV1212 hEF1a:Tal14
Golden Gate
TAL14 High Sensors
Midi Prep
Double input low sensor
GG Donor for TC
NOTES
hEF1a-TAL14 is still giving us troubles (the gel doesn't quite make sense)

Retrieved from "http://2014.igem.org/Team:MIT/Notebook"