Team:MIT/Notebook

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NOTES<br>
NOTES<br>
hEF1a-TAL14 is still giving us troubles (the gel doesn't quite make sense)
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)
 +
2 might have worked
 +
Sent for sequencing.
 +
Turns out, didn't work. eYFP missing.
 +
 +
July 25
 +
SS
 +
Did midipreps. Very strange distribution of concentrations:
 +
LilrB2 1: ~1200
 +
LilrB2 2: ~700
 +
PirB 7: ~4000
 +
PirB8: ~300
 +
Digesting midipreps just in case.
 +
LilrB2: XbaI, ApaI
 +
PirB: ApaI
 +
 +
LilrB2's are weird. Running undigested controls.
 +
LirB2 DNA is contaminated!
 +
Correct band pattern is most definitely there. Which means all parts: hEF1a promoter, LilrB2 gene and backbone are there.
 +
AG
 +
Tissue culture
 +
 +
 +
July 26
 +
SS
 +
Run DNA for gel extraction. Also look to see if other, verified DNA contaminated or not.
 +
Pick colonies fusions
 +
Transform
 +
Transforming with L2 and L3 DNA (L24, L34).
 +
 +
 +
July 27
 +
SS
 +
Minipreped a gazillion fusions.
 +
4/5 cofilins seemed to work. Had 3 fragments. Means at least 2 of the goldengated parts are present. Will digest with ApaI tomorrow to check if 3rd is present.
 +
PirBs didn't seem to work, will pick more colonies.
 +
LilrB2 didn't work
 +
Will pick pEXPR_LilrB2 colonies.
 +
AG
 +
Tissue culture
 +
 +
July 28
 +
SS
 +
Cofilin 1, 3, 4, 5 worked, need to do another digest.
 +
LilrB2:TCSG4 1, 4, 5 might have worked. Strange extra band on top. Will do different digest.
 +
LilrB2: YFPL none worked. Will miniprep more.
 +
PirB:TCSG4 none worked.
 +
PirB:YFP none worked.
 +
Cofilin worked! Sending all 4 for sequencing, 'cuz PCRs and mutations, ya know?
 +
LilrB2:TCSG4 did not work. Will have to miniprep more.
 +
 +
Made glycerol cell stocks of all the cofilins, but due to some mistake, the labels got erased. So, unless they all worked, I'll just re-transform one that worked.
 +
Making glycerol cell stocks of pEXPR_hEF1a: LilrB2 (L2 and L3)
 +
 +
July 29
 +
midipreped pEXPR_hEF1a:LilrB2.
 +
All the eYFP:Cofilin worked! LRed today.
 +
Did more golden gates.
 +
Picked more colonies, too.
 +
Inoculated midi culture for ggDonr
 +
 +
July 30
 +
More fusions digested!
 +
New goldengates of fusion proteins transformed
 +
New LR of hEF1a:YFPCofilin transformed
 +
LR of TREt:YFPCofilin done.
 +
 +
July 31
 +
SS
 +
LTs might have worked. Did digest again with different enzymes.
 +
Did digest with some other interesting ones: LT12 and PT11 and PT12.
 +
Transformed TREt:TEVpCofilin LRs
 +
Picked hEF1a:TEVpCofilin LRs
 +
Picked pENTR:YFPCofilin
 +
Picked more LY and PY from new goldengates
 +
Sent LT and PT for sequencing
 +
 +
LA
 +
Transfected LilrB2 for membrane localization
 +
Gel verification of LilrB2 and PirB PCR products ran
 +
 +
August 1
 +
SS
 +
Miniprep PY, LY, pENTR_TEVpCofilin
 +
Verify Minipreps
 +
Transform hEF1a:YFPCofilin
 +
Pick TREt:YFPCofilin Colonies
 +
All 4 pENTR_TEVpCofilin worked!
 +
The others didn't work. Given the lack of blue colonies, I'm thinking backbone religation/only one part present (probably YFP). Need to figure out something else.
 +
 +
Sequencing:
 +
LilrB2TCSG4 seems to have worked! 11 and 13 might have point mutations (high G content, unlikely). Will proceed with 12.
 +
PirBTCSG4 seems to have worked, too! 13 seems to have a point mutation (high G content, unlikely). Will proceed with 11.
 +
 +
August 2
 +
SS
 +
Transform all LRs (hEF1a:PT, hEF1a:LT, hEF1a:TC, TREt:TC)
 +
Pick hEF1a:YC colonies
 +
Miniprep TREt:YC if it worked. Otherwise, wait for Brian. Worked.
 +
Midiprep inoculate TREt:YC
 +
Digest LilrB2TCSG4 with NcoI and PstI seperately.
 +
Digest TREt:YC with ApaI
 +
 +
TREt:YFP_Cofilin 1, 2, 4 verified. Will midiprep 2.
 +
Ran PT (pENTR_PirBTCSG4) to make sure it isn't contaminated).
 +
The gel for PT had an extra band, but the sequencing results show that the TCSG4 is present and goes into the PirB region. That means everything is in order.
 +
 +
 +
August 3
 +
SS
 +
Took pEXPR TREt YC midiprep out of incubator
 +
Took pEXPR_hEF1a:YC miniprep out of incubator
 +
Took plates out of the incubator
 +
plates dried out. Picked colonies for TREt-TC
 +
 +
August 4
 +
SS
 +
Miniprep pEXPRs (TREt TC, hEF1a YC)
 +
Check pEXPRs (TREt TC, hEF1a YC)
 +
Inoculate Midipreps
 +
Pick colonies for hEF1a LT, PT, TC
 +
Digest TREtTC with ApaI Cutsmart
 +
Digest hEF1aYC with BsrGI 2.1
 +
Digest LT with BglI 3.1
 +
Digest TC with PvuII with 3.1
 +
Digest LT11 with BlpI with CS
 +
Give up on hEF1aYC. Won't be using it anyway.
 +
 +
pEXPR_TREt:TEVpCofilin is wrong. Gives weird patterns. Need to pick again.
 +
pENTR:LilrB2TCSG4 is wrong. Big chunk is actually missing. Need to pick again.
 +
 +
August 5
 +
SS
 +
Miniprep pEXPR (hEF1a: PirBTCS, hEF1a: TEVpCofilin)
 +
Verify hEF1a: PirBTCS
 +
Miniprep pENTR LilrB2TCS
 +
Verify pENTR LilrB2TCS
 +
Midiprep inoculate hEF1a: PirBTCS
 +
Pick more TREt:TEVpCofilin
 +
Plate more TREt:TEVpCofilin if possible.
 +
 +
Digest pEXPR_hEF1a: PirBTCSG4VP16 with BsrGI, buffer 2.1
 +
Digest pENTR_LilrB2:TCSG4VP16 with XhoI +XbaI, buffer CS
 +
Order: hEF1a PT 1, 2, 3, 4, LT16, 17, 18, 19
 +
 +
August 6
 +
SS
 +
Miniprepped more pENTR_LilrB2TCS
 +
Verify hEF1a: PirBTCS. Else pick more colonies. Picked more colonies
 +
Verify pENTR LilrB2TCS. Including LT16. Did not work
 +
 +
Verify pEXPR_TREt:TEVpCofilin. Worked!
 +
 +
Midiprep inoculate TREt:TEVpCofilin if it worked.
 +
Midiprep inoculate hEF1a: PirBTCS if it worked. Didn't work
 +
Sent pENTR_LilrB2 (04 2) for sequencing again...
 +
 +
Digest pEXPR_hEF1a: PirBTCS (1, 3, 4) with BsrGI, 2.1
 +
Digest LT (21-25) with XbaI, XhoI, CS
 +
Digest LT16 with NcoI, XhoI, CS
 +
Digest pEXPR_TREt:TEVpCofilin with BsrGI, 2.1
 +
 +
hEF1a: PirBTCS did not work. Need to pick more
 +
 +
 +
August 7
 +
SS
 +
Miniprepped more hEF1a: PirBTCSG4. Some worked!
 +
Midiprep inoculating.
 +
Also cell stock
 +
PCR'd LilrB2 and YFP. LilrB2 band is too low. YFP didn't work.
 +
Midiprepped TREt:YFPCofilin and TREt:TEVpCofilin.
 +
 +
 +
August 8
 +
SS
 +
Midiprep pEXPR_PirBTCSG4VP16. The midiprep concentration was low, around 16. Probably because the culture didn't grow very well.
 +
Miniprep inoculating again.
 +
Digest any YFP fusion minipreps we have to check for what segments are present and what went wrong.
 +
Dilute midipreps.
 +
 +
 +
August 12
 +
AG
 +
hEF1a:PirB-TCS-Gal4VP16 midiprepped
 +
LilrB2 pENTR sent for sequencing
 +
 +
August 14
 +
AG
 +
pENTR LilrB2:
 +
Blunt end cloning of gBlock
 +
 +
pEXPR hEF1a:PirB-YFP
 +
Transform
 +
 +
 +
August 22
 +
AG
 +
Miniprepped pENTR-LilrB2 (1-6)
 +
Ran digests.
 +
They look good.
 +
Sent LilrB2 1 for full sequencing. Waiting till sequence verified for making cell stock.
 +
 +
August 23
 +
SS
 +
Transformed pEXPR_hEF1a:LilrB2 and pEXPR_TREt:LilrB2
 +
 +
August 24
 +
Ss
 +
Got sequencing results back. The pENTR is sequence verified!
 +
Picked colonies for pEXPRs.
 +
 +
August 25
 +
Miniprep LRs
 +
Digest LRs
 +
Also digest pEXPR_hEF1a: PirBYFP
 +
PCRing at 53 degress, 40 seconds.
 +
Digesting with:
 +
pEXPR_hEF1a: LilrB2 XhoI, CS
 +
pEXPR_TREt: LilrB2 XbaI, CS
 +
pEXPR_hEF1a: PirB_YFP ApaI, CS
 +
 +
Both LilrB2 pEXPRs worked!
 +
 +
August 26
 +
SS
 +
Midis didn't work. Had ~10ng/ul concentration.
 +
So, taking the cell stocks and throwing them out.
 +
 +
August 27
 +
SS
 +
The pENTR_PirB_YFP did not actually have the YFP insert.
 +
 +
August 28
 +
SS
 +
Picked pENTR LilrB2_YFP/TCS colonies
 +
Put pEXPR_LilrB2 midi culture in incubator (~8:45pm)
 +
 +
August 31
 +
SS
 +
Transformed pEXPR hEF1a:LilrB2 and TREt:LilrB2 (10pm)
 +
Digesting pENTR LilrB2YFP and LilrB2TCS
 +
LilrB2YFP: PstI                  3.1
 +
LilrB2TCS: XhoI + XbaI    CS
 +
 +
Looks like LT4 worked. Will send for sequencing (M13F and R)
 +
The LYs don't seem to have worked. May be a problem with the YFP PCRs.
 +
 +
September 1
 +
SS
 +
Inoculated midi for pEXPR(hEF1a/TREt):LilrB2
 +
 +
September 2
 +
Midiprepped pEXPR(hEF1a/TREt):LilrB2
 +
Concentrations around 1ng/ul.
 +
pENTR_LilrB2TCS sequencing results came. Both components are there.
 +
 +
September 3
 +
SS
 +
LR'd LilrB2TCS (hef1a, tret)
 +
 +
September 4
 +
SS
 +
Transformed LRs
 +
Tried midiprepping pEXPR_hef1a/tret:LilrB2. The vacuum manifold wasn't working.
 +
 +
 +
</body>
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Revision as of 09:55, 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)

Native Receptor



June 15
AG, LA SS
Grew pENTR_LilrB2, pENTR_PirB

June 16
Liquid cultures didn't grow
SS
New liquid cultures made
AG, LA
NEGEM and High School presentations completed

June 17
Liquid culture still didn't grow BUT we figured out why
SS
Made new cultures and incubate them overnight
Made ggplates
LA
Prepare for a practice HEK293 transfection with Nelson
AG
Send ggDONR for sequencing
Split HEK293 cells

June 18
Liquid culture STILL didn't grow
New plates + old ggDONR grows and turn blue
We think something is wrong with the plates
Primers for fusion have arrived
LA
Prepared media for HEK293 in preparation for transfection transfection

June 19
Plates from our transformation grew blue and white colonies
PirB2 didn't grow any white colonies
SS
Liquid culture - to make stock - of ggDONR from troubleshooting experiment
golden gated again
AG
Made liquid culture of GGDonr and pENTR_L1_LilrB2_L2 for miniprep

June 20
SS
Took liquid cultures out of incubator and put in fridge.
AG, LA, SS
Attended NEGEM 3.1

June 22
LA
Transformed GoldenGated pENTR_L1_LilrB2_L2, pENTR_L1_PirB_L2
Plated
SS
Made cell stock of GGDonr and pENTR_L1_LilrB2_L2 (from previous GoldenGate)
Miniprepped GGDonr and pENTR_L1_LilrB2_L2 (from previous GoldenGate)
AG
Problem with HEK-293 cell culture - culture another plate

June 23
AG
Transformed Golden Gate product:
positive control didn't grow any colonies
blue and white colonies on LilrB2
only white colonies on PirB
picked colonies
SS
Digested previous pENTR_LilrB2 and GGDonr (BssSI, 3.1)
Could not figure out how to make the gel imaging station to work. But...
Gel did not fluoresce under UV, not even the hyperladder.

June 24
Liquid cultures from the second golden gate grew
SS
Liquid cultures miniprepped and DNA waiting to be verified

June 25
AG
Ran verification gels of the products of the minipreps - one very bright band for each lane
We suspect enzyme (BssSI) didn't work - DNA was super coiled. Old enzyme has been thrown out.
Split HEK293 cells. Prepare for practice transfection

June 26
SS
Digested LilrB2, PirB and GGDonr (again). EcoRI this time
AG
Practice transfection (fluorescent protein - 1 plasmid)

June 29
AG
Plated 3rd GG pENTR_LilrB2, pENTR_Pirb (Treatment group did control.)
Flow cytometry of practice transfection - high efficiency
Prepare for more complex transfection - 2 plasmids
June 30
LA
Picked colonies
cultures grew
found inactive cofilin plasmid
need to verify sequence and see if it has been used before
if suitable - will order today
started designing rest of fusions on geneious
AG
Transfection - 2 plasmids

July 1
SS
Miniprepped DNA
Restriction digested for verification
cofilin order on the plasmid page

July 2
SS
Ran gels of yesterday's digest
Did digests again on select pENTRs
40ul total volume instead of 20ul, same amount DNA (500ng)
Ran gel
AG
Flow cytometry - lower efficiencies (keep practicing).
Train team members in tissue culture

July 3-2
SS
Imaged gel that we ran yesterday

July 6
SS
Transformed LR Product (pEXPR hEF1a:PirB and hEF1a:LilrB2)
Picked colonies (3 each) and grew up overnight in liquid culture
Mini-prepped and nano-dropped
AG
Split HEK293 cells

July 7
LA
PCRs to make fusions started
50 ml liquid cultures for midi prep in incubator

July 8
AG
pEXPRs midiprepped
LR product digested and gel ran
LA
PCRs for fusions in progress
Digesting LR's
Hef1a:LirB2 BsrGI
Hef1a:PirB ClaI

July 9
Sequencing results received for pENTR
LilrB2 is correct
PirB: Qsite ligation with the C terminus gblock in pENTR
New PirB colonies picked
Restriction digest on pEXPR LilrB2 repeated because we suspected quality of digestion
LilrB2 = 1900 bps
PirB = 2500 bps (won't work because pENTR is not what we thought it was)
AG
Split HEK293 cells. Start 3 plate series so that splitting would occur every day.

July 10
SS
Digested pENTR_PirB with EcoRI.
Made primers for PirB, LilrB2, Cofilin fusion to YFP.
LA
Sent pENTR_PirB for sequencing for M13 F and R primers.
Sent pEXPR_hEF:LilrB2 from yesterday that seemed to work (L2, L3) for full sequencing.

July 11
LA
Send pEXPR hEF1a:LilrB2 and pENTR PirBfor sequencing (L2, L3)

July 14
AG
TEVp-cofilin fusion to be ordered as a gBlock
Split HEK293 cells.
LA
Sequencing results returned and analyzed
pEXPR hEF1a:LilrB2 didn't give us a good enough read - we're sending those out again
pENTR L1_PirB_L2 worked
SS
L1_PirB_L2 LRed

July 16
SS
Received primers - started PCRs
Done:
LilrB2 (eYFP)
PirB (eYFP)
LilrB2 (TCS-Gal4VP16)
PirB (TCS-Gal4VP16)
To be done:
Cofilin (eYFP)
Ran verification gel on PCR products

LA
streaked cofilin plasmid DNA
Sequencing results returned for LilrB2 pEXPR
LilrB2 insert is there: LilrB2 pEXPR verified
"No Priming" for second read in both samples?
AG
PirB pEXPR transformed
Split HEK293 cells.

July 17
SS
PirB LR colonies picked and liquid cultures prepared
LilrB2 PCRs repeated under 2 different (higher) annealing temperatures
Remaining PirB PCR product ran and band with the correct size DNA to be extracted
Gel with PCR products imaged
Expected sizes:
LilrB2: 2,000
PirB: 2,500

July 18
SS
PirB PCR bands extracted
Concentrations very low
around 5 ng/ul
PirB pEXPRs miniprepped
Restriction digest on PirB pEXPRs run
LA
PirB PCRs run again
LilrB2 PCRs done at higher annealing temperatures
Cofilin plasmid colonies picked
AG
Tissue culture

July 19
pEXRP_hEF1a:PirB 2 seems to give the correct band pattern,
but the bands are slightly lower than they should be.
Ran extraction gel for PCR products: HL1. LY65, LT67, LY68, LT70, PY, PT
Turns out the gel was upside down, so all the products diffused. Need to do all the PCR's again
SS
Cofilin plasmid miniprepped. Concentrations ~600ng/ul
Re-did PirB PCR's, Did Cofilin PCR. LilrB2 PCR's need to be redone
Current cell glycerol stocks: pENTR_PirB (verified), pENTR_LilrB2 (verified), pEXPR_hEF1a:LilrB2 (verified),
pEXPR_hEF1a: PirB (unverified), Cofilin gene in backbone.
AG
Tissue culture

July 20
SS
Did PCR's of LilrB2
Ran gel of all the PCR's
Ladder, LilrB2 YFP 65, LilrB2 YFP 68, LilrB2 TCSG4 67, LilrB2 TCSG4 70, PirB YFP, PirB TCSG4, Cofilin YFP

July 21
SS
Miniprepped new pEXPR_hEF1a: PirB (4-8) ~200ng/uL
Digested new pEXPR_hEF1a: PirB
Ran Gel
Looks like they all worked!
Sent pEXPR_hEF1a: PirB 2 (from saturday) for sequencing. Will not be good quality (did not have enough DNA)
Transform pEXPR_hEF1a: PirB (4-8)
Plate pEXPR_hEF1a:LilrB2 (cell stock)
AG
Tissue culture

July 22
SS
Liquid culture for hEF1a:(Receptor) midiprep grown up - to be midiprepped tomorrow
eYFP-cofilin golden gate transformed

July 23
SS
Miniprep pEXPR_hEF1a: LilrB2/PirB:
A couple came out to have around 700ng/ul. Others were around 400ng/ul
Midiprep Culture:
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.
Also inoculated pEXPR_hEF1a: LilrB2.
Midiprep party tomorrow!
eYFP-Cofilin:
Plate had lots of white colonies and a few blue ones. Not expected. Expecting a lot of backbone re-ligation. Picked 10.
Will miniprep tomorrow.
AG
Tissue culture
July 24
SS
Miniprepped eYFP:Cofilin colonies
Ran digest gel (PstI, ApaI, Cutsmart) 2 might have worked Sent for sequencing. Turns out, didn't work. eYFP missing. July 25 SS Did midipreps. Very strange distribution of concentrations: LilrB2 1: ~1200 LilrB2 2: ~700 PirB 7: ~4000 PirB8: ~300 Digesting midipreps just in case. LilrB2: XbaI, ApaI PirB: ApaI LilrB2's are weird. Running undigested controls. LirB2 DNA is contaminated! Correct band pattern is most definitely there. Which means all parts: hEF1a promoter, LilrB2 gene and backbone are there. AG Tissue culture July 26 SS Run DNA for gel extraction. Also look to see if other, verified DNA contaminated or not. Pick colonies fusions Transform Transforming with L2 and L3 DNA (L24, L34). July 27 SS Minipreped a gazillion fusions. 4/5 cofilins seemed to work. Had 3 fragments. Means at least 2 of the goldengated parts are present. Will digest with ApaI tomorrow to check if 3rd is present. PirBs didn't seem to work, will pick more colonies. LilrB2 didn't work Will pick pEXPR_LilrB2 colonies. AG Tissue culture July 28 SS Cofilin 1, 3, 4, 5 worked, need to do another digest. LilrB2:TCSG4 1, 4, 5 might have worked. Strange extra band on top. Will do different digest. LilrB2: YFPL none worked. Will miniprep more. PirB:TCSG4 none worked. PirB:YFP none worked. Cofilin worked! Sending all 4 for sequencing, 'cuz PCRs and mutations, ya know? LilrB2:TCSG4 did not work. Will have to miniprep more. Made glycerol cell stocks of all the cofilins, but due to some mistake, the labels got erased. So, unless they all worked, I'll just re-transform one that worked. Making glycerol cell stocks of pEXPR_hEF1a: LilrB2 (L2 and L3) July 29 midipreped pEXPR_hEF1a:LilrB2. All the eYFP:Cofilin worked! LRed today. Did more golden gates. Picked more colonies, too. Inoculated midi culture for ggDonr July 30 More fusions digested! New goldengates of fusion proteins transformed New LR of hEF1a:YFPCofilin transformed LR of TREt:YFPCofilin done. July 31 SS LTs might have worked. Did digest again with different enzymes. Did digest with some other interesting ones: LT12 and PT11 and PT12. Transformed TREt:TEVpCofilin LRs Picked hEF1a:TEVpCofilin LRs Picked pENTR:YFPCofilin Picked more LY and PY from new goldengates Sent LT and PT for sequencing LA Transfected LilrB2 for membrane localization Gel verification of LilrB2 and PirB PCR products ran August 1 SS Miniprep PY, LY, pENTR_TEVpCofilin Verify Minipreps Transform hEF1a:YFPCofilin Pick TREt:YFPCofilin Colonies All 4 pENTR_TEVpCofilin worked! The others didn't work. Given the lack of blue colonies, I'm thinking backbone religation/only one part present (probably YFP). Need to figure out something else. Sequencing: LilrB2TCSG4 seems to have worked! 11 and 13 might have point mutations (high G content, unlikely). Will proceed with 12. PirBTCSG4 seems to have worked, too! 13 seems to have a point mutation (high G content, unlikely). Will proceed with 11. August 2 SS Transform all LRs (hEF1a:PT, hEF1a:LT, hEF1a:TC, TREt:TC) Pick hEF1a:YC colonies Miniprep TREt:YC if it worked. Otherwise, wait for Brian. Worked. Midiprep inoculate TREt:YC Digest LilrB2TCSG4 with NcoI and PstI seperately. Digest TREt:YC with ApaI TREt:YFP_Cofilin 1, 2, 4 verified. Will midiprep 2. Ran PT (pENTR_PirBTCSG4) to make sure it isn't contaminated). The gel for PT had an extra band, but the sequencing results show that the TCSG4 is present and goes into the PirB region. That means everything is in order. August 3 SS Took pEXPR TREt YC midiprep out of incubator Took pEXPR_hEF1a:YC miniprep out of incubator Took plates out of the incubator plates dried out. Picked colonies for TREt-TC August 4 SS Miniprep pEXPRs (TREt TC, hEF1a YC) Check pEXPRs (TREt TC, hEF1a YC) Inoculate Midipreps Pick colonies for hEF1a LT, PT, TC Digest TREtTC with ApaI Cutsmart Digest hEF1aYC with BsrGI 2.1 Digest LT with BglI 3.1 Digest TC with PvuII with 3.1 Digest LT11 with BlpI with CS Give up on hEF1aYC. Won't be using it anyway. pEXPR_TREt:TEVpCofilin is wrong. Gives weird patterns. Need to pick again. pENTR:LilrB2TCSG4 is wrong. Big chunk is actually missing. Need to pick again. August 5 SS Miniprep pEXPR (hEF1a: PirBTCS, hEF1a: TEVpCofilin) Verify hEF1a: PirBTCS Miniprep pENTR LilrB2TCS Verify pENTR LilrB2TCS Midiprep inoculate hEF1a: PirBTCS Pick more TREt:TEVpCofilin Plate more TREt:TEVpCofilin if possible. Digest pEXPR_hEF1a: PirBTCSG4VP16 with BsrGI, buffer 2.1 Digest pENTR_LilrB2:TCSG4VP16 with XhoI +XbaI, buffer CS Order: hEF1a PT 1, 2, 3, 4, LT16, 17, 18, 19 August 6 SS Miniprepped more pENTR_LilrB2TCS Verify hEF1a: PirBTCS. Else pick more colonies. Picked more colonies Verify pENTR LilrB2TCS. Including LT16. Did not work Verify pEXPR_TREt:TEVpCofilin. Worked! Midiprep inoculate TREt:TEVpCofilin if it worked. Midiprep inoculate hEF1a: PirBTCS if it worked. Didn't work Sent pENTR_LilrB2 (04 2) for sequencing again... Digest pEXPR_hEF1a: PirBTCS (1, 3, 4) with BsrGI, 2.1 Digest LT (21-25) with XbaI, XhoI, CS Digest LT16 with NcoI, XhoI, CS Digest pEXPR_TREt:TEVpCofilin with BsrGI, 2.1 hEF1a: PirBTCS did not work. Need to pick more August 7 SS Miniprepped more hEF1a: PirBTCSG4. Some worked! Midiprep inoculating. Also cell stock PCR'd LilrB2 and YFP. LilrB2 band is too low. YFP didn't work. Midiprepped TREt:YFPCofilin and TREt:TEVpCofilin. August 8 SS Midiprep pEXPR_PirBTCSG4VP16. The midiprep concentration was low, around 16. Probably because the culture didn't grow very well. Miniprep inoculating again. Digest any YFP fusion minipreps we have to check for what segments are present and what went wrong. Dilute midipreps. August 12 AG hEF1a:PirB-TCS-Gal4VP16 midiprepped LilrB2 pENTR sent for sequencing August 14 AG pENTR LilrB2: Blunt end cloning of gBlock pEXPR hEF1a:PirB-YFP Transform August 22 AG Miniprepped pENTR-LilrB2 (1-6) Ran digests. They look good. Sent LilrB2 1 for full sequencing. Waiting till sequence verified for making cell stock. August 23 SS Transformed pEXPR_hEF1a:LilrB2 and pEXPR_TREt:LilrB2 August 24 Ss Got sequencing results back. The pENTR is sequence verified! Picked colonies for pEXPRs. August 25 Miniprep LRs Digest LRs Also digest pEXPR_hEF1a: PirBYFP PCRing at 53 degress, 40 seconds. Digesting with: pEXPR_hEF1a: LilrB2 XhoI, CS pEXPR_TREt: LilrB2 XbaI, CS pEXPR_hEF1a: PirB_YFP ApaI, CS Both LilrB2 pEXPRs worked! August 26 SS Midis didn't work. Had ~10ng/ul concentration. So, taking the cell stocks and throwing them out. August 27 SS The pENTR_PirB_YFP did not actually have the YFP insert. August 28 SS Picked pENTR LilrB2_YFP/TCS colonies Put pEXPR_LilrB2 midi culture in incubator (~8:45pm) August 31 SS Transformed pEXPR hEF1a:LilrB2 and TREt:LilrB2 (10pm) Digesting pENTR LilrB2YFP and LilrB2TCS LilrB2YFP: PstI 3.1 LilrB2TCS: XhoI + XbaI CS Looks like LT4 worked. Will send for sequencing (M13F and R) The LYs don't seem to have worked. May be a problem with the YFP PCRs. September 1 SS Inoculated midi for pEXPR(hEF1a/TREt):LilrB2 September 2 Midiprepped pEXPR(hEF1a/TREt):LilrB2 Concentrations around 1ng/ul. pENTR_LilrB2TCS sequencing results came. Both components are there. September 3 SS LR'd LilrB2TCS (hef1a, tret) September 4 SS Transformed LRs Tried midiprepping pEXPR_hef1a/tret:LilrB2. The vacuum manifold wasn't working.

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