Team:BostonU/Notebook
iGEM Notebook
Made with Benchling
Project: Protocols
Authors: Kamila Drezek
Dates: 2016-05-19 to 2016-10-09
Thursday, 5/19/16
Preparing Reagants
Saturday, 5/21/16
LB Broth (1L)
20 grams per liter
LB Agar (1L)
40 grams per liter
Prepare two liter containers of each- one per antibiotic (Cam and Carb)
Autoclave for 1 hour
Preparing chloramphenicol stock (1000x)
Final concentration of 1x chloramphenicol should be 25 ug/ml
Stock concentration of 1000x chloramphenicol should be 25 mg/ml
Prepare 50 ml of stock to aliquot into microcentrifuge tubes by measuring 1.25 g of chloramphenicol and ~50ml of 200 proof ethanol in a 50 ml Falcon
Filter chloramphenicol through 20 um syringe filter
Preparing Plates
20ml agar per plate
Innoculate Vector 1721
5 ml of LB+carb
Stab cell stock
Grow overnight at 37C shaker
Sunday, 5/22/16
Preparing Cell stock of 1721
Box Name- iGEM 2016 Source Box
Freezer Number- 3
Shelf- 3
Vector 1721 is miniprepped
See protocol sheet
Vector 1721 is digested (300ul digest)(1-2hrs)
24000 ng digestion of DNA
30 ul buffer (cold room)
9ul of BpsI (-80C, small green box)(incubation in 37C)
101 ul of water
Digestion Clean-Up of Vector 1721
See PCR cleanup protocol
Note- vector was eluted in 50 ul of EB not 30 ul.
Dilution of Oligos (ideal final concentration is 100uM)
Short Cut-
300 ul of TE for values ~25- ~35 nmol
200 ul of TE for value ~15 - ~24 nmol
Note- Oligo 12, 13, 14, 17, 18, and 20 was vortex without liquid initially
Annealing of pGEX oligos
See protocol
Annealing time- 2 minutes
dilute oligos to 1ng/ul
Ligation of 1721 and insert
Note- diluted annealed oligos named- pGEX#A where # is the number of the construct
24 reactions
139.2 ul of water
24 ul of T4 Ligase
24 ul of T4 Ligase Buffer
26.4 ul of Vector
Transformation
See protocol
Plates put in incubator at 7:09 PM- Desired pick up time is 10:09
Monday, 5/23/16
pGEX plates were pulled from incubator at 10:15
Growth and density of all samples exceeded that of the negative control
Tuesday, 5/24/16
Picked two colonies per plate and grew in 4ml of LB+carb in 37C shaker.
Created stab plate for each colony.
Wednesday, 5/25/16
Minipreping colonies
Notes: During the miniprep samples from colony 10 were contaminated
Test Cut
4 ul DNA
1 ul 2.1
.25 of ul each enzyme (Bsp1 and Nco1)
4.5 ul water
Run digestion for at least 15 minutes
Test Cut Results
All colonies appeared with proper bands at 1.1 kb and 4.2kb and no bands at 440 bp. We decided to send only the first colony from each set in for sequencing
Thursday, 5/26/16
Sequencing results are in
All sequences with the exceptions of pGEX1-1, pGEX14-1, and pGEX 15-1 were successful.
All sequences reamining sequences were then grown up for cell stock
Note: While pGEX 6-1 sequenced properly the tube containing its reference appeared to have been contaminated and pGEX 6-2 will be sent in for sequencing to reverify
Sequencing sent in
pGEX: 1-2, 6-2, 14-1, 14-2, 15-1, 15-2, 10-1, 10-2
Restriction digest of 2490 and DC16 with MluI and NruI
5ul buffer3.1
6ul DNA
1.5ul Nru1
1.5ul Mlu1
36ul water
Prerequisites to subvectors which will be used to create one plasmid with Cre and both Flps
Subvectors to be made have homology with each other, allowing for easy gibson assembly with no PCR (promoters and ERT2 - modified estrogen receptor - have repeat regions)
Digestion of DC16 yielded 3 bands rather than the expected 2. Culprit may be unreliable cell stock.
Ligate 2490 and DC16 to create 2534 (10ul)
See Ligation Calculator
4.2 ul DI water
1.0 ul DC16 (insert)
2.8 ul 2490 (vector)
1 ul T4 Ligase Buffer
1 ul T4 Ligase
Construct 2534 and a negative control are transformed
Tissue Culture Room
Prepping hood for trannsfection
Remember to clean the hood down with 70% ethanol
Aspirate all media from the plate to aid in the trypsin function
Kidney cells are stored in T75 flask
After aspiration perform a PBS wash
Phosphate Buffer Solution- a salt water solution (~5ml wash)
Trypsin is used remove kidney cells from the plate.
Add 2.5ml of Trypsin to the T75
Dont shoot directed at the cells, let the trpysin run over them.
Addition of Medium will neutralize trypsin
Add 7.5 ml of media to plate
And tritrate (Pipette up and down)
To improve cell count- spin cells down then respend them
Spin at 300 RCF for 2-5 minutes
To resuspend- tap the hood with the 50ml falcon tube
Add back 10 ml of media and tritrate
Performing a cell count
Add 10 ul of cell solution to a cell counting slides
Divide the cell count concentration by 2
Actual Cell Counts
~2.5 Million cells per ml
Magic Number for transfection:
200,000 cells per ml
For each T75- 20 ml of solution
For desired concentration of cells-
Add 4.8 ml of cell concentrate
Add 55.2 ml of media
Continuing the cell line
Note:
Split/Passage: diluting cells in the same vessel
Split the parent cell line
Friday, 5/27/16
pGEX Cells moved to Stock
1-13, 16-20
Sequencing results
6-2 and 1-2 are perfect
pGEX 14 ans 15 failde to sequence again
Transformation for Construct 2534
No significant growth in the positive- less than what appeared in the negative -> failure
Digest a different aliquot of DC16 at a low concentration
Need 4ng of DNA with DNA at 45ng/uL so need 89 uL of DNA; will do 100 ish uL digest
89 uL DNA
1.5 uL MluI
1.5 uL NruI
10 uL Buffer 3.1
No water
Ligation of dc16 insert and 2490 vector
2.7 ul of vector
1.7 ul of insert
3.5 ul of water
1 ul of T4 Ligase
1 ul of T4 Ligase Buffer
Digestion of pSb1A3 and pBS1C3
40 ul of DNA
5 ul of Cutsmart BUffer
1.5 ul of EcoRI
1.5 ul of PstI
2 ul of Water
After digestion treat witl DnpI for 15 minutes
PCR Clean- up and elute in 30 ul of EB
Gibson of pSb1A3 (2533) and pSb1C3 (2531)
CONSTRUCT NAME 2531
Mass Vec =25
Ratio = 3
V A B C D W
SIZE 2000 1200 600 0 0
CONC 5.6 10 10 1 1
VOL 4.5 4.5 2.3 0.0 0.0 -1.2
CONSTRUCT NAME 2533
SIZE 2000 1200 600 0 0
CONC 23 10 10 1 1
VOL 1.1 4.5 2.3 0.0 0.0 2.2
Both the Gibson Assembly Transformation and the Ligation Transformation are over 10kb
See Protocol
Stable Line Integration of 2494-2496
See Protocol for Stable Line Transfection
Construct 900- Piggy back transpose
Cell status preceeding transfection
Low cell count ~30-40% confluences
Thaw out 2 tubes of PEI
Use an incubator in a BL2 room
Note: We are making 4x the number of cells than what is required
Mixes
DNA part
.78 ul of transposase
3.2 ul of DNA
.15 M NaCl (3ml per tube)
PEI part
2ml of PEI
.15 M NaCl ( 10.5 ml)
Add 4 ml of PEI part to DNA part
Transfection
Don't pour directly onto the- shoot away from the cells
Saturday, 5/28/16
Create cell stock of pGEX10-1
MIniprep of 14-3/4/5 15-3/4/5 and 2534-1/2
Test Cut Results
pGEX 14-3, pGEX 15-5, 2534-2 => sent to sequencing
Cell inspection preceeding puromycin selection
~90% confluence
Puromycin Selection
4ul of concentrated puromycin - 2ug per ml to each flask
Wait 10 days for total transient plasmids are diluted outs.
Sunday, 5/29/16
Sequencing Results
pGEX 15-5 can be moved to cell stock
pGEX 14-3 failed
2534 failed
Sent Sequencing
pGEX 14-4, pGEX 14-5
Digestion
2531
4 uL DNA
4 uL DI
1 uL CutSmart
0.5 uL EcoRI and AgeI
2533
4 uL DNA
4 uL DI
1 uL CutSmart
0.5 uL EcoRI and AgeI
DC16 midiprep
1 uL DNA at 1:10 dilution
7 uL DI
1 uL Buffer 3.1
0.5 uL MluI and NruI
DC16 miniprep
2 uL DNA
6 uL DI
1 uL Buffer 3.1
0.5 uL MluI and NruI
dc16 Primers
BP0165, BP0162 reverse
BP0198, BP0176 forward
dc56 Primers
BP0165, BP0130 reverse
BP0038, BP0188 forward
pGEX 14-4 and pGEX14-5 moved to cell stock pending sequencing
Tuesday, 5/31/16
Phosphorylating and Annealing Oligos for pGOP
Moved Construct 2531 to cell stock
Grow up more 2531 for ligation
Because of Chloramphenicol's effect on growth, we will grow this up for 24 hours
Oligos are at 1000x concentration- dilute to 1x (~1ng/ul)
Picked more colonies from 2533 and grew up overnight
Wednesday, 6/1/16
Miniprep 2531 (grown again due to low inital concentration) and 2533 (initial sequencing was incorrect)
Digest 2531 with BbsI (50 uL total)
30 uL DNA
5 uL Buffer 2.1
2 uL BbsI
13 uL DI water
Ligate oligos with 2531 to create pGOP1-20 (10uL total)
See protocol
0.4 uL 2531 (BbsI)
1.3 uL annealed oligos
6.4 uL DI water
1 uL T4 Ligase Buffer
1 uL T4 Ligase
All pGOP plasmids were transformed and plated (25 ul) with Control
Test Cut 2533 with EcoR1 Age1
2533-3/4/5 were sent in for sequencing
Prepped for creation of comp cells for tomorrow
Mix 25g PEG8000 with 30ml water and disolve over night
Thursday, 6/2/16
PIck 2 colonies for each pGOP
Miniprepped dc16 and dc56
Prepared 200ml TSS Solution
dc 16 test cut with MluI and NruI
expected bands: 10723, 1172
dc 56 test cut with AgeIHF and EcoRIHF
expected bands: 6700, 1000
Primer 0170 for 2533 failed and this impeded the sequencing
Preparing Top10 comp cells
First OD (optical density) measurement: 0.2 (time: 2:15 p.m.)
Second OD measurement: 0.34 (time 2:45 p.m.)
Third OD measurement: 0.419 (time 3:05 p.m.)
Fourth OD measurment: 0.486 (time 3:35 p.m.)
Friday, 6/3/16
miniprepped pGOP plasmids
test cut pGOP plasmids with Bbs1 and Spe1
grew up more _____ for midiprep
Saturday, 6/4/16
Picked three colonies of pGOP 20 (3, 4, and 5)
Grew up pGOP 20-3/4/5
Miniprep 2533-7/8/9
Sunday, 6/5/16
Miniprep 20-3/4/5
Test Cut for 2533 and pGOP 20 addition colonies
Monday, 6/6/16
Miniprep pGOP20-R1/R2/R3
Test cuts for pGOP20-R1/R2/R3
Cell Stock pGOP 1-19
Grew up DC16-1 and DC56-1 for cell stock
Sent pGOP 20-3/4/5 and 2533-7/8/9 for sequencing
Diluted pGEX 1-20 and pGOP 1-19 to 50 ng/uL
Made a liter of LB + Carb
Nanodropped pGEX 1-20 and pGOP 1-19
Inoculated pGEX16-1 due to low concentration
Midiprep for Transfection
Prepared 150 mL of LB + Carb with pCJH2
Prepared 150 mL of LB + Carb with T40
Prepared 150 mL of LB + Carb with pBW363
Tuesday, 6/7/16
Plan for transfection of pGEXs and pGOPs to be performed on Thursday
Miniprep of pGOP12, pGEX16, DC174 and DC100 (both DCs will be used as positive controls transfection on Thursday)
Midiprep of pBW363 (Blank), T40 (dCas9), pCJH2 (BFP transfection marker), DC16 and DC56
Evaporated supernatant
Wednesday, 6/8/16
Cell stocked pGOP 20
50ul Restriction digest of 2490 and DC16 with MluI and NruI to create 2534
5ul buffer3.1
6ul DNA
1.5ul Nru1
1.5ul Mlu1
36ul water
incubate 1 hr in 37C
Got 3 bands so threw out digest and are now investigating our enzymes to see if there has been contamination
Stocked midiprep DNA with 1x TE
Grew up T40 for further minipreps
Prepared HEK293 plates for tomorrow's transfection
Add 250ul of 200000cells/ml cell solution to each well on 48 well plate
Thursday, 6/9/16
Transfected HEK cells
Miniprepped T40
Diluted pCJH2, pBW363, and T40 for transfection
Checked and rediluted pGEXs and pGOPs for transfection
note- pGEX11 is contaminated
pGEX7 is too low of concentration
Friday, 6/10/16
Checked transfection plates under a microscope
all worked; however, Cre showed leakiness
Resent DC16-1 for sequencing due to QuintaraBio sequencing faillure
Passaging HEK293 cells
Monday, 6/13
Minipreppred pGOP1-20 (inoculated in Cam last night)
Cut DC16 and 2490 with MluI and NruI
8 uL DC16 or 11uL of 2490
1.5 uL MluI
1.5 uL NruI
5uL buffer 3.1
34 uL water or 31 uL water (2490)
Run Gel Extraction on 2490 and DC16
Bands
DC16
10723
1172 - WANTED BAND
2490
10590 - WANTED BAND
227
Ligation of 2490 and DC16 to make 2534
Ligations were transformed and plated
Tuesday, 6/14
Diluted primers for PCR reaction of pGPX1 (2531)
PCR GFP, BFP and mRuby
Digested pGOPs 3,5,6,8,10,14,15,19 and pGPX1 with Mlu1-HF and Age1-HF in preparation of making pGOPs with GFP, BFP and mRuby
Miniprep 2531
Picked colonies for 2534
Transfected HEK cells with different concentrations of the 8 pGOPs given above, to test whether the concentration of pGOPs in the cell was causing high levels of basal expression)
Transfected transfection marker, T40 (dCas9) pGEXs, Blank plasmid and pGOP. pGOP at 62.5, 30 and 7.5 ng
PCR digested GFP, BFP and mRuby with Mlu1-HF and Age1-HF
Wednesday, 6/15
Minipreppred T40 (was running low)
Ligated GFP, BFP and mRuby into pGOP 3,5,6,8,10,14,15,19. Transformed onto Cam plates
Thursday, 6/16
Ran FACs on transfection from Tuesday
PCR the silencer out of DC100 (has UAS and pG5) to put into pGOPs and pGPX6 with PrA4 and PrA4r
Digested DC 100 and prepared annealed oligos o83-o122 to prepare _________
Friday, 6/17
Miniprepped Transformed plasmids from 6-15-16
PCR for GFP, BFP, and mRuby
Sent plasmdis in for sequencing
Sequence for 2534B-4 is verified as functioning and will be moved forward with cloning
Monday, 6/20
Cell Stocked plasmids for BFP, GFP, and mRuby that sequenced properly
Miniprepped plasmids
Picked colonies for mRuby pGOP 10,14, and 15
Tuesday, 6/21
Cell stocked BFP pGOP 15
Picked colonies for GFP pGOP 8, GFP pGOP 19, mRuby pGOP 10, mRuby pGOP 15, and mRuby pGOP 19
Miniprepped plasminds from 6-20-16 (mRuby pGOP 10-3, 10-4, 14-3, 14-5, 14-6, 15-3)
Sent mRuby pGOP 14-3/4/5 and BFP pGOP 19-2 for sequencing
Passaged new HEK cells to P7
Made 2 new bottles of D5 Media (D5= 5%FBS)
500ml of DMEM base
25ml FBS (alloquoted in fridge across hallway from TC room)
5ml L-Glut
5ml P/S
5ml sodium pyruvate
Wednesday, 6/22
Cell stocked picked colonies from 6/21/16 (GFP pGOP 8, GFP pGOP 19, mRuby pGOP 10, mRuby pGOP 15, and mRuby pGOP 19).
Thursday, 6/23
Gel extract PrA4/4r (silencer)
Digested DC100 with NheI and dropped in annealed oligos with ligation to make pDOP 1-20 (backbone from DC100 with gRNAs dropped in)
sent pGOP 6-9 back in for sequencing - suspected error in gRNA target site
Monday, 6/27
ligate and transform pDOP 1-20 (DC100 and annealed oligos)
inoculate pGPX1 so we can make pGPX2 and 3
Tuesday, 6/28
Prepped for transfextion
Picked pDOP1-20
Gel extracted pGPX1
Wednesday, 6/29
ligate and transfrom pGPX2 and pGPX3 (pGPX1 MluI Age1 with PrA1/1r and PrA2/2r Mlu1 and Age1)
miniprep pDOP 1-20
Test Cut pDOP1-20 with NheI-HF and StuI
Transfected pGOP 3,5,6,8,10,14,15,19 of iRFP, BFP, GFP, and mRuby
Thursday, 6/30
PIcked pGPX2 and pGPX3 colonies
Reannealed-phosphorylated- ligated - and transformed pDOPs
recut pDOPs
Friday, 7/1
Miniprep of GPX 2-1/2/3 and GPX 3-1/2/3
Test-cut GPX 2 and 3
Picked colonies and grew up pDOP 1-4; 6-11; 14-20
Ran FACS (flow cytometry)
Did not test BFP due to wrong transfection marker
PCR clean up of 2485; 2487; and 2489 with NotI and Cip
Gel extraction of 2484, 2486, 2488, and 2534 digested with AscI and NotI
Saturday, 7/2
Miniprep and test cut pDOP 1-4; 6-11; 14-20 colonies 1 and 2
digested with NheI and PstI
Vectors pGPX2 and pGPX3 are sequenced verifeid. pGOP 66,34, and 26 could not be sequenced properly (quintara error)
Reenoculate pGEX and GFP pGOP 3, 5, 6, 8, 10, 14, 15, 19 gh54
Sunday, 7/3
Miniprep pGEX and GFP pGOP 3, 5, 6, 8, 10, 14, 15, 19
Gibson and transform pREC 1, 2, 3
Monday, 7/4
Passage HEK cells
Inoculate pREC 1, 2, 3
Tuesday, 7/5
Miniprep pREC 1, 2, 3
Enoculate BW1720
Digested BW1720, and BW1942-43 with BbsI
Digested BW758, and BW762-65 with AgeI and EcoRI
Digested DC 100 with NheI and Cip
Digested pGPX1-4 with Xbha and Bbsi
Transfected HEK cells for gRNA orthogonality experiment
Wednesday, 7/6
Miniprep pGEX, GFP pGOP, and mRuby pGOP 6& 14
Digested 1720 with AgeI and EcoRI
Redigested 2485, 2487, and 2489 with NotI and Cip
Ligated and transformed pBEX1-24 pPV1,5-8
Retransformed pGEX6 and 14 pGOP26,34,66,74
Made LB + Carb plates
Thursday, 7/7
Picked colonies for pBEX 1 - 24, pGEX 6 and 14, GFP and mRuby pGOPs 6 and 14, adn pPVs 1, 5, 6, 7, 8
Miniprep pGEX 1-4
Ran FACs
Grew more 2534
Friday, 7/8
Monday, 7/11
Grew up pPV7 and pBEX 10, 12, 13, 17, 18, 22
Growing more 2534
Gibsoned pREC1, 2, and 3
Passaged HEK cells
Tuesday, 7/12
Cell Stock pPV7 and pBEX 10, 12, 13, 17, 18, and 22
Digested pGPX2, mRuby2, and pGOP 1, 2, 4, 6, 7, 9, 11, 12 13, 16, 17, 18, and 20
mRuby2 and pGOPs digested with MluI and AgeI
pGPX2 digested with BbsI
Ran on a gel and gel extracted
Wednesday, 7/13
Miniprep pGPX2, 1720, 1942-44
Digested pGPX2, 1720, 1942-44 with ________________
Re-digested pGPX2 (bbs1, extract 3kb band) ,mRuby2(mlu1, age1; extract 700bp band) ,and pGOP21 (mlu1,age1, extract 3kb band)
ran on gel and gel extracted
Thursday, 7/14
Ligating and transforming
pGOP 21 22 24 27 29 31 32 33 34 36 37 38 40 41 42 44 47 49 51 52 53 56 57 58 60 61 62 64 66 67 69 71 72 73 76 77 78 80
Digesting
1270 758 763 765 with AgeI and EcoRI
Circuit Designs Digesting
Part1 AscI NotI
Part2 AscI NotI
Part3 AscI NotI
Part4 AscI NheI
Dest EcoRI NotI
Circuits (P1-P2-P3-P4)
pBEX1-pBEX6-pBEX15-pBEX24
pBEX1-pBEX10-pBEX19-pBEX20
pBEX5-pBEx18-pBEX19-pBEX16
pBEX5-pBEX2-pBEX15-pBEX8
Test cut 2578, 2579, 2580 (formely pRECs)
Friday, 7/15
Miniprep 2578, 2579, and 2580 3-7 & testcut
2578-2 and 2579-1 were sequenced verified adn moved to cell stock
Minipreped 1716 pDest
Digested pDest with EcoRI and NotI
Religated pPV1, pPV6, and pPV8
Picked new pGOP colonies for GFP only
Saturday, 7/16
Miniprepped new GFP clonies
Minprepped 2580-8 through 2580-17
Transformed pCir1 pCir2 and pCir3
Sunday, 7/17
Test Cuts for 2580 aditional colonies
Test Cuts for pPV1, pPV6, and pPV8 additional colonies
Test Cuts for pGOP GFP additional colonies
Monday, 7/18
Test Cuts for pCIR1, pCir2, and pCir3
Test Cuts for pGOp1 and 2 under BFP and mRuby
Split HEK cells 1:10
Created two 48 well plates at 200,000 cells/ml for transfection of BFP pGOP constructs
Created two T175plates with 25ml of 100,000cells/ml for stable integration
Tuesday, 7/19
Midiprep BW361, BW465, BW471, BW 474, 2578, and 2579
Digested pBEX 19 with AscI and NotI
Gel extracedracted pBEX 19
Digested pCir 1-3, 1-1, 3-2, 3-3 with Not1
Transfected BFP
Wednesday, 7/20
Thursday, 7/21
Performed FACs on the BFP transfected cells. Tried to create the data in MEFL but had trouble with getting the channel names to work with the software.
Split HEK cells to P16. Expanded them into large T175 plate
Transfected the HEK cells in T175 with 2578 and 2579. Brought media volume up to 50ml to do so
Friday, 7/22
Saturday, 7/23
added puromyocin to the two stable line integrations of 2578 and 2579. 2ug per ml so we added 10ul of puromyocin to 50ml of media in the flask
many more cells ttoday than yesterday
Sent in a sample of the pGOP41 used in the previous transfection to troubleshoot what went wrong and why that particular gRNA was not working with BFP
Sunday, 7/24
Split the stable line integration HEK cells of 2578 and 2579 each into two new T175 flasks because they were too crowded. Added puromyocin to the fresh media.
Transformed (GFP) pGOP 26, 27, 32, 33, 38, 40
(BFP): pGOP 41, 42, 43, 44, 46, 47, 49, 51, 52, 53, 56, 57, 58, 60
(mRuby): pGOP 61, 62, 64, ,66, 67, 69, 71, 72 73, 76, 77, 78, 80
Monday, 7/25
PIcked colonies from all transformed GFP pGOPs and less successful BFPs and mRubys
Prepped overnight ligation for the BFPs and mRubys
due to miscommunication, the ligation mix was made incorrectly but we went ahead anyway
Tuesday, 7/26
Digested pBEX 1,2,5,6,16,19 (AscI and NotI except 16 which was AscI and NheI) and 1716 (EcoRI and NotI) and pGOP68 and 35 (MluI and AgeI)
did not extract the pGOPs or 1716
extracted the rest
Inoculated pBEX 1,6,16,19, and 1716 and pGPX2 (so we could drop in the new oligos)
Transformed DNA from overnight ligation
made Cam plates
Wednesday, 7/27
Transformation did not work
Digested 1716 (EcoRI and NotI) and pGOP68 and pGOP35 (MluI and AgeI)
gel extracted
annealed multimerized oligos
digested more pGPX
Diluted all the DNA for the transfection
Transfected HEK cells with the "mini circuit" (1902/1903, pGOP1, pGOP22, BW390 (constituitive cre) and 2196
also transfected pGOP22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 to test all of the GFPs. we did not have pGOP 21 and we did not have pGOP 40. We did not have pGEX11 to transfect so pGOP31 should not work.
organized our DNA boxes
Thursday, 7/28
Friday, 7/29
Digested more BFP and mRUBY
26ul pGOP55 30ulpGOP64
1.5 of MLU1-HF &Age1-HF 1.5 of Mlu1-HF &Age1-HF
5 cutsmart 5 cutsmart
20 h2o 16 h2o
ligated to create pGOP41, 51, 82, 83 to se what wasnt working with our cloning by testing our T4 ligase vs Wong labs t4 ligase
Testcut pCir1-6, -7, -8, -9, -10, -11, -12, -13, -14, -15, pCir3-4 with Acs1 Not1-HF
Cell stocked pGOP38-4, pGOP42-1, pGOp43-1, pGOP44-1, pGOP26-5, pGOP27-4, pGOP32-4, pGOP32-4, pGOP33-4, pGOP60-1, pGOP64-1
performed FACs
Split Deboki's cells for her and took a t175 plate at 1:10. (passage again on monday)
Moved the stable line integrations to T50 plates. added 2ul of puromycin to 10ml of media
Picked colonies for GOP 82, and 41, 51 (no colonies for 83), pCIR1s that Ben made (picked 10 colonies)
Met with Leidy to discuss what we are doing wrong with our ligation and transformation protocol.
Re did digesestion of BFP and mRuby from pGOP 43 and 64, respectively. (age1 mlu1)
Redigested pgpx2 to drop in the oligos.
Religating fluorescence into regular pGOPs
Saturday, 7/30
Picked colonies of pGOP 66-1*(failed),-2, 67-1*(failed),-2, 71-1*(failed),-2, 73-1*(failed),-2, 76-1*(worked),-2, 46-1*(worked),-2, 47-1*(failed),-2., 51-1*(failed),-2, 52-1*(failed), 53-1*(worked),-2, 56-1(failed)*,-2, 49-1*(failed),-2, 69-1*(failed sequencing reaction),-2, 41-1*(worked),-2,-3 -4,-5, 51-1,-2,-3,-4,-5,-6, 40-1,-5
pGOP 81-1,-2, 82-1*,-2, 83-1,-2,84-1,-2, 85-1,-2, 82-0* (-0 means grew up day before), 86-1,-2,87-1,-2,88-1*,-2, 89-1,-2, 90-1,-2, 83-0
pCIR1 -16,-17-,-18, -19,-20*,-21,-22,-23, -24,-25*,-26*,-27*, Ben's1, Ben's2*
(starred**** ones sent for sequencing)
Sunday, 7/31
miniprep of all of the colonies picked yesterday
Ran test cuts of all of the colonie picked
Monday, 8/1
split HEK cells 1:10 from large flask into small flask.
let stable line of cells grow for another day.
Grew up.... pGEX11, pGEX12, pGEX13, pGEX17, pGEX18, pGEX20, pGOP6(x2),pGOP21, pGOP22, pGOP23, pGOP24, pGOP25, pGOP26, pGOP27, pGOP28, pGOP29, pGOP30, pGOP31, pGOP32, pGOP33, pGOP34, pGOP35, pGOP36, pGOP37, pGOP38, pGOP39, pGOP42, pGOP43, pGOP44(x2), pGOP45(x2), pGOP50, pGOP54, pGOP55(x2), pGOP59, pGOP68, pGPX2
Tuesday, 8/2
(Will and Marisa) miniprepped pGEX11, pGEX12, pGEX13, pGEX17, pGEX18, pGEX20, pGOP6(x2),pGOP21, pGOP22, pGOP23, pGOP24, pGOP25, pGOP26, pGOP27, pGOP28, pGOP29, pGOP30, pGOP31, pGOP32, pGOP33, pGOP34, pGOP35, pGOP36, pGOP37, pGOP38, pGOP39, pGOP42, pGOP43, pGOP44(x2), pGOP45(x2), pGOP50, pGOP54, pGOP55(x2), pGOP59, pGOP68, pGPX2
Grew up pGOP41, pGOP46, pGOP53, pGOP71, pGOP76 for cell stock
Split the stable line HEK cells into new T75 flasks (p18)
Wednesday, 8/3
suspended the crRNA gBlocks oligos in 1xTE
Digested pGPX2 with BBs1
DIgested pGOP76-1 and pGOP 41-1 for mRuby and BFP
Transformed pGOPs 57, 58, 61, 62, 66, 77, 78, 81, 84, 85, 86, 87, 88, 89 using Leidys transformation protocol
use Ben's Top Ten cells, let cells sit on ice
add 10ul of 5x KCM to ligation mix
add 30ul of DI H2O to ligation mix
add all 50 ul to cells
plated 95ul of cells onto 1/2 plate
Sent sequencing pGOP 47-1(worked), 49-1(worked), 51-2(worked), 52-1(worked), 56-2 (failed), 67-1(worked), 69-1(worked), 71-1(FAIL), 72-1(worked), 73-1(resendfor seuquencing), 83-0(resend for sequencnig)
Cell stocked pGOP41, pGOP46, pGOP53(need to throw awayb/c miscommunication), pGOP76 (throw away b/c miscommunication)
Thursday, 8/4
(Marisa) Picked 2 colonies for pGOPs 57, 58, 61, 62, 66, 77, 78, 81, 84, 85, 86, 87, 88, 89
(Kami) Split wildtype cells 1:20 to P19
(Jeffery) dimerized guides 1, 3, 8, 13 in various configurations with each other (1-3, 3-1, 8-13, 13-8)
this involved PCR and a gel extraction
Designed decoders
(Will) Grew up pGOP 47, 49, 51, and 82-0 for cell stock
Due to a miscommunication error, we had to retransform and plate pGOP52-1, pGOP53-1, pGOP67-1, pGOP69-1, pGOP71-2, pGOP72-1, pGOP73-1. We did this with our typical transformation protocol.
Friday, 8/5
(Marisa and Will) Miniprepeed pGOPs 57, 58, 61, 62, 66, 77, 78, 81, 85, 86, 87, 88, and 89
Due to a miscommunication error, miniprepped pGOP 47, 49, 51, and 82-0
(Will) Gibsoned
pCir 2 using 1716, A, B, C, D
pCir 2 using 1716 Part1/2, Part 3/4
pCir 2 usng 1716, Decoder 2
pCir 4 using 1716, lambda, Part 3/4
pCir 1 using 1716, alpha, Bex19
pCir 1 using 1716, Part 1/2, Bex19, Bex16
pCir 1 using 11716 Bex 1, Bex6, Bex19, Bex16
pCir 3 using 1716, Bex5, Bex2, Bex19, Bex16
(Kami) Split stabel line (2578 and 2579) 1:10 in T75
(Rachel) Test-cut pGOPs 57, 58, 61, 62, 66, 77, 78, 81, 85, 86, 87, 88, and 89
Saturday, 8/6
(Rachel) digested pGPX2 (5mg) with 3ul of BbsI. Double digested - too low of concentraiton to proceed with ligation
Sunday, 8/7
(Will) grew up
Monday, 8/8
(Jeffery) Digested pgpx2 with bbsi
(kami) made WT Hek plates with Yash and split WT and 2578 and 2579 integrated cells 1:10
(will) cell stocked pGPX2
(Will) Test cut the pCir gibsons with Asc1 and Not1
(Will) transformed pGOP 84. 86, 87, 88, 89, 90 with a ligation using Leidy's protocol
transformed pgOP47, 49, 51, 82-0 without a ligation using Leidy's protocol
Tuesday, 8/9
(Rachel) Growing pCir4_2_4 from stab plate as sequencing indicated it worked!
(Rachel) Picked colonies from pGOP 81,84,86,87,88,89,90
(Rachel) made more LB + Cam broth
(Kami) autoclaved tips and agar
(Marisa and Kami) made carb plates
(Jeffrey) performed overhang extension PCRs on G-blocks (that make decoder) to build Circuit 2
Wednesday, 8/10
(Marisa and Will) miniprep pGOP 81,84,86,87,88,89,90
test cut with speI and BbsI
(Marisa) Cell Stock pGOP 47, 49, 51, and 82; pCir 4
miniprep pCir 4
(Jeffrey) Digested Bex 19 and 16
Thursday, 8/11
(Kami and Marisa) grew up BW 363, 390, 391; pSB1C3; and T40 for midiprep
(Kami) split WT, 2578, 2579 HEK cells into T175
(Jeffrey) Picked Colonies for 87 and 88
Friday, 8/12
(Marisa) Midiprep BW 363, 390, 391, T40, pSB1C3
(Jeffrey) MIinprepped and test 87 and 88... picked colonies for 90. Transformed interlab study plasmids
Saturday, 8/13
(Marisa) Midiprep BW 363, 390, 391, T40, pSB1C3
Cell stock pGOP 87
(Marisa and Rachel) Gel extracted
(Jeffrey) Mini prep and test cut 90... Retransformed interlab device 1
Sunday, 8/14
(Marisa and Rachel) Cell stock Interlab study: Pos 1, Neg 1, Part 2-1, Part 2-2, Pos 2, Neg 2, Part 3-1, Part 3-2
Miniprep Interlab Study and pGOP 56-3/4/5, pGOP 83-5/6/7/8/9
(Rachel) Digested pGOPs 56 and 83 with SpeI and XmaI
(Marisa) Test Cut pGOPs 56 and 83
(Rachel) Ligate
Monday, 8/15
(Jeffrey and Marisa) picked and grew up colonies: pGOP 57-1, 61-1/2/3, 62-1/2/3, 66-1/2/3, 77-1/2/3, 78-1/2/3, 80-1/2/3, 88-1/2/3/4/5, 90-1/2/3/4/5
(Kami and Marisa) Diluted and nanodropped
(Kami) made 6 plates for transfection. 3 with WT, 2 with stable line integrations of 2578 and 2579 each.
Tuesday, 8/16
(Marisa) miniprepped pGOP 57-1, 61-1/2/3, 62-1/2/3, 66-1/2/3, 77-1, 78-1/2/3, 80-1/2/3, 88-1/2/3/4/5, 90-1/2/3/4/5
(Will) Digested Marisa's minipreps
(Kami and Rachel) 7:30 pm transfected HEK cells to test the multimerization and the circuit
for the circuit. Transfected 1/8 of 250ng of each component. (2 pgops made up 1/8 of the ng transfection and the extra was filled with Luis's pSB2C3 blank)
(Kami And rachel) 2 hours after transfection, we added 4OHT/Absistic acid/Rapalog to appropriate wells
Wednesday, 8/17
Thursday, 8/18
(Kami and Rachel) prepared the transfected cells for FACS, and ran facs
(Kami) Cells stocked stabel lines of HEK 2578(m150) and 2579 (m151)
trypsonize cel
neutralize with media
spin cells down
remove media and break up pellet.
resuspend pellet in 90%FBS 10%DMSO (1ml per cryotube)
one T75 makess 2-3 cell stocks
(Marisa) Made LB Broth
inoculated T40 and BW363 in falcon tubes
turn LB Broth into LB + Carb broth
Friday, 8/19
(Kami) made cam agar and broth
miniprep pGOP 2, 6, 83-11/12/13/14/15
(Marisa) made Cam plates
Cell stocked pGOP 57, 61, 80, 88, and 90
inoculated 1mL of each T40 and BW363 sampled into flasks with 150 mL LB + Carb
Digested pGOPs 83 with SpeI and BbsI for test cut
Saturday, 8/20
(Marisa) Midiprep three of both T40 and BW363
Monday, 8/22
(Marisa) Grew up pGOP 82
(Jeffrey) Grew up pGOP 83-14
(Jeffrey) took OD 600 of the interlab and diluted cells down to .02
(Will) PCR'ed the pCMV fro the iGEM registry
(Will) Gibsoned and transformed the CMV PCR product into pGEX
(Marisa) made 96 well plate for InterLab Study
250 uL DPBS and 2uL cells per sample
(Kami Rachel) prepared HEK plates for transfection at 200,000 cells
Tuesday, 8/23
(Marisa) Cell stock pGOP 83
Grew up BW 1945, 1946, 1947, and 1948
(Will) Miniprep pGOP 82 ad 83
(Jeffrey) Picked colonies from GFP and BFP under igem CMV
(Jeffrey) Transformed and plated interlab bacteria
(Kami Rachel Marisa) made trasfection DNA mixes and transfected the HEK cells we plated yesterday
Added drugs to inducible cells 2hrs after transfection
Drugs are located in pink box labeled "Small molecules"
Rapalog= AC heterodimerizer (1000x)
Absistic Acid (1000x)- vortex before use to get rid of percipitant
4OHT (1000x)
Wednesday, 8/24
(Marisa) Miniprepped and nanodropped pCIR 4, pBex 16 pBEX 19, CMV BFP-1, CMV GFP-1/2, 1945, 1946, 1947, 1948
Grew up Bex 16 and Bex 19
(Jeffrey) Digested 1945, 1946, 1947, and 1948 with BbsI
Grew up pPV 1/5/6/7/8, 1942, 1943, 1944, and 1716
(Marisa) Grew up pCir 4 (x2), DC 56, BW 390, and BW 391
Made LB + Carb
Thursday, 8/25
(Rachel and Kami) Ran FACS
(Kami) split HEK293 and stable line cells into big flasks
(Marisa) miniprep pPV 1, 5, 6, 7, 8, pBEX16, 19(x3), BW1942, BW1943, BW1944, BW1716
midipreped DC56, BW390, BW391, pCir4(x2)
(Will) cut for gel extraction 1916, 1945, 1947, 1948 with BBs1
gel extracted those as well
(Will) grew up iGEM CMV for cell stock
(Rachel) annealed the mutated oligos
Friday, 8/26
(Jeffrey and Marisa) Midiprep BW 2286, BW 2287, and pCir 4 (x2)
Cell stock CMV GFP-1
(Rachel) picked two colonies for pGOP 101-150
(Will) Gel extracted pBEX 16 and 19
pBEX 16 digested with AscI and NheI
pBEX 19 digested with AscI and NotI
PCR overhang extension with sequences 37 and 56
Saturday, 8/27
(Kami, WIll, Rachel) Performed minipreps for Mismatches and additional multimerized operators pGOP 101-150
Monday, 8/29
(Will) Test Cut for Mismatches
(Jeffrey) Attempted overhang extension PCR ... Failed
Tuesday, 8/30
(Jeffrey) Attempted overhang extension PCR ... Failed
(Jeffrey) Completed Interlab Study
(Marisa) PCR GEX plasmids for CAM transfer
(Will) Gel extracted PCR
Wednesday, 8/31
(Marisa) Digested pGOP 109, 110, 112, 114, 134,l 135, 136, 138
(Marisa) Grew up pGEX 7, pGEX 8, pGPX1,
(Jeffrey) Attempted overhang extension PCR ... Failed
(Jeffrey) Test Cut 109, 110, 112, 114, 134,l 135, 136, 138
Thursday, 9/1
(will) pcr mruby and bfp to drop into pgops
(Jeffrey) MInipreped pgop 80-90 for multiplecolors , bexs for circuits, gexs f
(Marisa) Passaged HEK cells
Made 2L of LB Broth, 2L of LB+Carb broth, and 2L of LB + Cam agar for plates
Ligated and transformed pGEX 3, 5, 8, 10, 15, and 19 into Cam (with BW 1721 -n330 backbone)
Grew up T4o (x2), BW 363, GEX 5, GEX 15, pCir 4 (x2), and GPX 2
(Rachel) autoclaved epi tubes and tips
Ligated and transformed BW 1945-1949
Friday, 9/2
(Marisa) Made LB + Cam plates
(Jeffrey) Resuspended cells grown for midiprop and stored in the cold room
(Rachel) Prepared and ran cells for FACS
(Will) Annealed oligos for multimerized pGOP 3
Saturday, 9/3
(Jeffrey) transformed mismatched pGOP 101-150
(Marisa and Jeffrey) grew up GEX 3, 5, 8, 10, 15, 19, BW 1721, 1945, 1946, 1947, 1948, and GOP 29
Sunday, 9/4
(Jeffrey) picked and grew up colonies from pGOP101-150
miniprep of GEX 3, 5, 8, 10, 15, 19, BW 1721, 1945, 1946, 1947, 1948, and GOP 29
Digested BW1945, 1946, 1947, 1948
(Marisa) Cell stock GOP 110, 112, and 114
Digested GOP 29(MOP) with EcoRI and PstI
PCR GEX 3, 5, 8, 10, 15, 19 with PrA6 and PrA6r
PCR clean up BW 1945, 1946, 1947, 1948 and MOP
Made and ran gel for GEX 3, 5, 8, 10, 15, 19 and BW1721
(with Kami) gel extracted GEX 3, 5, 8, 10, 15, 19, and BW 1721
Digested GEX 3, 5, 8, 10, 15, 19 and BW 1721 with EcoRI and PstI
PCR clean up GEX 3, 5, 8, 10, 15, 19, and BW 1721
Monday, 9/5
(Marisa) Cell stock GOP 101-103; 105-108; 111; 115-134; and 146
Miniprep pGOP 101-103; 105;108; 111; and 115-122
Ligate and transformed pGEX 3, 5, 8, 10, 15, and 19 into pSB1C3 backbone
(Will) Miniprep pGOP 123-134 and 146
(Kami Rachel) seeded 8 48 well plates at 100,000cells for transfection on wednesday
Tuesday, 9/6
(Marisa) picked two colonies of GEX 3, 5, 8, 10, 15, and 19 (now termed 103, 105, 108, 110, 115, and 119) from transformation 9/5
(Jeffrey) Performed overhang extension pcr to build the decoder for circuit 2
(Jeffrey) transformed the 18T entry vectors for circuit 5
Wednesday, 9/7
(Marisa) miniprep GEXs 103, 105, 108, 110, 115, and 119
Test cut with AseI
picked two colonies for 18T-terminator circuits
(Rachel, Marisa and Kami) transfected HEKcells
Thursday, 9/8
(kami Marisa) passaged HEK cells
(jeffrey) performed a gibson to build circuit 2
(jeffrey) test cuts for 18T entry vecotrs for circuit 5 indicated low digestion efficeincy and the circuits needs to be retransformed
FRIDAY 9/9
(Will) Ligated and transformed GEX 21-40 and GOP 151-168
(Will and Marisa) Grew up 3 colonies for GEX 21-40 and GOP 151-168
Saturday 9/10
(Will Jeffrey Marisa) Miniprepped GEX 21-40 and GOP 151-168
(Marisa) PCR GEX 3, 5, 8, 10, 15, 19, and BW 1721 with PrA6 and PrA6r (previous GEX 103, 105 108, 110, 115, 119 were not successful)
(Marisa and Will) gel extracted GEX 3, 5, 8, 10, 15, and 19 and BW 1721 (~400); will be insert for GEX 103, 105, 108, 110, 115, and 119
Sunday, 9/11
(Marisa) Digested GEX 103, 105, 108, 110, 115, and 119 and BW 1721 inserts with EcoRI-HF and PstI-HF
(Jeffrey) Picked colonies for 18T entry vectors and miniprepred circuit 2 for test cuts
(Will) Test cut GEX 21-40 and GOP 151-168
Monday, 9/12
(Marisa) picked four colonies for 18T g1, g17, g3, g8, and g13
Digested and test-cut 18T g1, g17, g3, g8, and g13 colonies 1 and 2 with BbsI and SpeI
(Marisa) Ligated and transformed inserts for GEX 3, 5, 8, 10, 15, and 19 into pSB1C3 backbone to create GEX 103, 105, 108, 110, 115, and 119
(Kami) made 8 48 well plates with HEK cells at 100,000 cells/ml
(kami) retransformed pGOP7 because it looked wierd when we transfected it. so going to pick one colony from this transformation to try to start fresh.
(Marisa) Made LB + Cam broth
Grew up pSB1C3, GOP 35, and GPX2 for midipreps
Grew up pSB1C3 and GOP 45 (x2) for minipreps
Tuesday, 9/13
(Marisa) picked two colonies for GEX 103, 105, 108, 110, 115, and 119
Midiprep pSB1C3, pGPX2, pGOP35
Miniprep GOP45 (x2), SB1C3, and g17 18T-1/2/3/4
made master mixes for transfection to take place on 9/14
picked and grew up a colony from GOP7 re-transformation
Added Carbacillin to plain LB Broth to make LB+Carb broth
Grew up Cir4 (x2), SB1C3 (x2), GEX5, GEX 15, GPX2, T40 (x2), and BW471
Wednesday, 9/14
(Marisa) miniprepped pGEX 103-1/2, 105-1/2, 108-1/2, 110-1/2, 115-1/2, and 119-1/2
Test cut GEXs with EcoRI-HF and PstI-HF
Test Cut g17 18T with BbsI and SpeI
(Marisa and Jeffrey) made 2L of plain LB broth
(Jeffrey) midiprep pSB1C3 (x2), T40(x2), and pCir4(x2)
(Kami Rachel) transfected Hek cells. with pGOPs diluted to 5ng/ul (not 50ng/ul) and add a bigger volume to the transfection mix.
Thursday, 9/15
(Marisa) Miniprep GEX 103-3/4/5/6, 105-3/4/5/6, 108-3/4/5/6, 110-3/4/5/6, 115-3/4/5/6, 119-3/4/5/6
Digest and test cut GEXs with EcoRI-HF and PstI-HF
(Marisa and Kami) passaged stable line HEK cells (Wild Type, 2578, and 2579) 1:20
(Jeffrey) Minipreped 1945-1948
Friday, 9/16
(Kami) Miniprep pGOP 159-1/2/3, 160-1/2/3, 169-1/2/3, and 170-1/2/3
(Will) Test cut pGOP 159, 160, 169, and 170
Saturday, 9/17
(jeffrey) ligated and transformed BEX34-38
Sunday, 9/18
(Marisa and Jeffrey) picked three colonies of GOP 151-158 and 161-168
(Jeffrey) Picked colonies for BEX37 Redigested 1945,1946,1948
(Jeffrey) Religated and transformed BEX 34,35,36,38
Monday, 9/19
(Marisa) Miniprep colonies 1,2, and 3 of GOP 151-158 and 161-168
(Jeffrey) PIcked colonies for BEX 34-38
(Kami) Made 6 hek plates at 100,000 (NOT USED)
(Will) Test cut the miniprepped GOPs 151-168
Tuesday, 9/20
(Marisa) Cell stock BEX 30, GOP 159, 160, 169
(Marisa) made 2L of plain LB broth
(Marisa) Diluted GOP 81-90 to 25 ng/ul
(jeffrey) Minipreppred bex 34-38 and test cut
(Jeffrey) Digested BEX 1,6,15,20, 30,31,32,33
(Jeffrey) digested parts vectors with bbsi for 3 input circuit
(Will) Sent in colony 1 of GOPs 151-168 that test cut correctly
Wednesday, 9/21
(Marisa and Rachel) Made LB agar (x2)
(Marisa) Cell stock pGOP 159, 160, and 169
(Marisa and Kami) Ligated GOP 153, 154, 157, 163, 165, 166, 168, 170, 162, and 167 with respective oigos
(rachel) transformed the pGOPs ligated above
(Kami) Made LB + Cam plates
(Marisa) Made LB + Carb plates
(Jeffrey) Gibson Cir2 and Cir 5 using BEX parts vectors
Thursday, 9/22
(Kami) picked 4 colonies for GOP 153, 154, 157, 162, 163, 165, 166, 167, 168, 170
(Jeffrey) Cell stock GOP 151, 152, 155, 156, 158, 161, 164
Miniprepped colonies 5-8 of GOP 153, 154, 157, 162, 163, 165, 166, 167, 168, 170
Test cut GOPs with BbsI and SpeI
Digested BEX 34-37 with AscI and NotI
Digested BEX 38 with AscI and NheI
(Kami) passaged HEK cells
(Will) Sent in colony two of GOPs 151-168 that did not sequence properly
Friday, 9/23
Cell stocked pGOP 154,
Sunday, 9/25
(Rachel) Ligated and transformed 1721 insert into MOP (GOP with biobrick cut out)
(Jeffrey) Gibson Circuit 2 and 5
Monday, 9/26
(Marisa) miniprepped four colonies of pGOP 104, 109, 135, 137, 144, 149, and 150
Test cut GOP (x3) of 91-100, GOP (x4) 104, 109, 135, 137, 144, 149, 150, 153, 154, 157, 162, 163, 165, 166, 167, 168, 170, and GOP (x3) 113, 135, 136, 138, 142, with BbsI and SpeI
(Marisa) Transformed Circuit 2 and Circuit 5
(Marisa) picked four colonies of MOP + 1721
(Will) Sent in GOP151 and 163 for sequencing
(Kami) made HEK plates
Tuesday, 9/27
(Marisa) PCR pGEX 3, 8, 15, and 19 and BW 1721
(Marisa and Will) Gel extracted pGEX 3, 8, 15, 19 and BW 1721
(Will) Digested GEX 3, 8, 15, 19 and BW 1721 with EcoRI-HF and PstI-HF
PCR cleanup of GEXs and BW1721
(Rachel) Ligated GEX 3, 8, 15, 19, and BW 1721 with MOP
(Marisa) Transformed GEX 3, 8, 15, 19 and BW 1721 (to GEX 103, 108, 115, and 119)
(Will) Cell stocked pGOP 151 and 163
Wednesday, 9/28
(Marisa) Test cut GOP 162, 163, and 167 colonies 5-8 with BbsI and SpeI
(Marisa) Cell stock 91-100, 104, 109, 113, 136, 137, 138, 142, 144, 149, 150, 153, and 157
(Marisa) miniprep GOP 112, 114, 135,
(Marisa) Replated Circuit 2 and 5
Thursday, 9/29
(Marisa) Cell stock pGOP 151 and 163
(Marisa) Replated Circuit 2 and Circuit 5
(Marisa and Rachel) ligated and transformed pGOP 142, 162, and 167
Friday, 9/30
(Marisa) picked 5 colonies of pGOP 167
(Kami) made 11 hek plates at 100,000cells/ul
(kami)Diluted pGOPs to 25ng/ul
Saturday, 10/1
(Marisa) Miniprep and test cut pGOP 167-1/2/3/4/5 with BbsI and SpeI
Sunday, 10/2
(Kami Rachel) Diluted DNA for transfection.
(Kami Rachel) Transfected hek cells with all the multimerized and mutated pGOPs
Friday, 10/7
(kami) made 7 hek plates at 100,000 for transfection on sunday and 1 hek plate at 200,000 cells/ml for transfection on saturday with WPI
(Rachel) prepared the transfection DNA mix for WPI
Saturday, 10/8
(Kami + Rachel) transfected cells for WPI testing the and/nor circuit
(Kami + Rachel) prepared all the DNA mixes for transfection tomorrow
Sunday, 10/9
(Kami) Transfected HEK cells with screen of mutated p GOPs, CMV compared to single and triple multimerized, and 4 analog circuits
