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Ryan Liang's notebook
From 2009.igem.org
8/13 Tranfections
1. Car1-FRB
2. hM4
3. SSF-YFP-hM4D-βPix
4. hM4D Act A Long
5. hM4D Act A Short
6. hM4D LPD Short
Transfection Efficiency
Car1-FRB: 42.4%
hM4: 42.4%
SSF-YFP-hM4D-βPix: 40.4%
hM4D Act A Long: 46.4%
hM4D Act A Short: 36.5%
hM4D LPD Short: 44.3%
Chemoattractant Dilutions
CNO [0nM, 10nM, 100nM, 1uM]
cAMP [0nM, 10nM, 100nM, 1uM]
fMLP [100nM]
Result: hM4D worked only
8/12
Transfections
1. GPR132
2. LPA1
3. OPRL1
4. Control
Transfection Efficiency:
GPR132: 41%
LPA1: 43.2%
OPRL1: 50%
Chemoattractant Dilutions
LPC: [0nM, 10nM, 100nM, 1uM]
LPA: [0nM, 100nM, 500nM, 1uM]
Orphanin FQ: [0nM, 1nM, 10nM, 100nM]
fMLP [100nM]
RESULTS: OPRL1 worked only
8/10
Transfections
1. DOR
2. DOR ACTININ
3. DOR ERM
4. DOR EZRIN
5. DOR KIFC
6. DOR VHEAD
7. hM2D
8. hM3D
9. hM3.2D
10. Control
Transfection Efficiency:
DOR: 40.9%
DOR ACTININ: 39.5%
DOR ERM: 47.5%
DOR EZRIN: 48.1%
DOR KIFC: 39.2%
DOR VHEAD: 32.1%
hM2D: 35.2%
hM3D: 51%
hM3.2D: 54.7%
Chemoattractant Dilutions
DADLE [0nM, 1nM, 10nM, 100nM]
CNO [0nM, 10nM, 100nM, 1uM]
fMLP [100nM]
Results: DOR, DOR ACTININ, DOR EZRIN, DOR KIFC, DOR VHEAD, and hM3.2D worked
8/6
Transfections
1. ADRA1A
2. EDG1
3. GRM2
4. GRM4
5. MTNR1A
6. OPRL1
7. VIB
8. Control
Chemoattractant Dilutions
Epinephrine: [0nM, 10nM, 100nM, 1000nM]
S1P: [0nM, 10nM, 100nM, 1uM]
Glutamate: [0nM, 10nM, 100nM, 1uM]
CNO [0nM, 10nM, 100nM, 1uM]
Melatonin: [0nM, 1nM, 10nM, 1uM]
Orphanin FQ: [0nM, 1nM, 10nM, 100nM]
Vasopressin [0nM, 10nM, 100nM, 1uM]
fMLP: [100nM]
8/5
Transfections
1. CCR7
2. DOR
3. DOR ERM
4. DOR EZRIN
5. DOR KIFC
6. DOR VHEAD
7. hM3
8. Hm3.2
9. Control
Transfection Efficiency:
CCR7: 45.2%
DOR: 62.8%
DOR ERM: 45.7%
DOR EZRIN: 40%
DOR KIFC: 35.2%
DOR VHEAD: 42.4%
hM3: 47.4%
hM3.2: 30.9%
Chemoattractant Dilutions:
MIP-3Beta (0ug/ml, 0.01ug/ml, 0.1ug/ml, 1ug/ml)
DADLE [0nM, 10nM, 100nM, 1uM]
CNO [0nM, 10nM, 100nM, 1uM]
RESULTS: Wildtype cells did not stain so our data is unreliable so we have to redo all these GPCRs and RASSLs. Of the results, it shows that CCR7, DOR, DOR EZRIN, DOR KIFC, DOR VHEAD, and hM3.2 migrated. DOR KIFC is a maybe. When we redo it, we will change the concentrations to 10x lower.
8.4
Transfections
1.ADRA1A
2.EDG1
3.GPR132
4.GRM2
5.GRM4
6.hM3
7.LPA1
8.MTNR1A
9.OPRL1
10.V1B
11. Control
Chemoattractant Dilutions
Epinephrine: [0nM, 10nM, 100nM, 1000nM]
S1P: [0nM, 10nM, 100nM, 1uM]
LPC: [0nM, 10nM, 100nM, 1uM]
Glutamate: [0nM, 10nM, 100nM, 1uM]
CNO [0nM, 10nM, 100nM, 1uM]
LPA: [0nM, 100nM, 500nM, 1uM]
Melatonin: [0nM, 1nM, 10nM, 1uM]
Orphanin FQ: [0nM, 1nM, 10nM, 100nM]
Vasopressin [0nM, 10nM, 100nM, 1uM]
fMLP: [100nM]
RESULTS: None, machine broke, redo 8/6
8/3
Transfections
1. AGTR1
2. AGTR2
3. B2AR
4. B2AR EZRIN
5. hM3.2
6. hM4
7. HTR1A
8. HTR2B
9. HTR7A
10. Rs1
11. Rs1.3
12. Control
Chemoattractant Dilutions:
Isoprenaline [0nM, 1nM, 10nM, 100nM]
CNO: [0nM, 10nM, 100nM, 1uM]
Zacopride: [0nM, 10nM, 100nM, 1uM]
Angiontensin II (0nM, 1nM, 10nM, 100nM)
Seratonin (0nM, 10nM, 100nM, 1000nM)
Glutamate [0nM, 10nM, 100nM, 1uM]
fMLP: [100nM]
7/31
Goal: Combine WT cells and GPCR cells in the same well/insert only if we can give WT RFP.
1. Transfection with DsRed2 and mCherry
2. Dye cells with Red Vybrant @ 0,1,2,5,15,20 minutes
DsRed2 and mCherry takes too long to show RFP (5+ hours)
Cells are dyed at the optimum amount by 15minutes.
7/29
Transwell
Test new and old stocks of CNO in Millipore and BD
Chemoattractant Dilutions:
fMLP [100nM]
CNO [10nM, 100nM] Old & New
7/28
Transfections
hM4 Transients in Millipore, BD, and Corning
Chemoattractant Dilutions:
CNO [0nM, 10nM, 100nM, 1uM]
fMLP [10mM]
RESULTS: BD is best
7/27
Site DIrected Mutagenesis of DOCK insert in pTOPO
Picked colonies for AB-SSF pDONR221-PL-Flag, PCR2.1 TOPO-ITSN, PCR2.1 TOPO-TUBA, pDONR221-LPD775-1250aa, AB-YFP pDONR221-PL-FLAG-YFP, and TOPO-LPD full
7/21
Transfections
1. ADRA1A
2. B2AR
3. B2AR EZRIN
4. HTR1A
5. HTR2B
6. GPR132
7. LPA1
8. Control
Chemoattractant Dilutions:
Epinephrine (0nM, 10nM, 100nM, 1000nM)
Isoprenaline(0nM, 100pM, 1nM, 10nM)
LPA (0nM, 100nM, 500nM, 1uM)
LPC (0nM, 10nM, 100nM, 1uM)
Serotonin (0nM, 10nM, 100nM, 1000nM)
7/20
Transfections
1. AGTR1
2. AGTR2
3. CCR7
4. GRM2
5. GRM4
6. MTNR1A
7. OPRL1
8. VIB
9. Control
Dilution Calculations
Angiontensin II (0nM, 1nM, 10nM, 100nM)
MIP-3Beta (0ug/ml, 0.01ug/ml, 0.1ug/ml, 1ug/ml)
Vasopressin (0nM, 10nM, 100nM, 1uM)
Orphanin FQ [0nM, 1nM, 10nM, 100nM]
Glutamate [0nM, 10nM, 100nM, 1uM]
Melatonin [0nM, 1nM, 10nM, 1uM]
7/15
Transfections
1. AGTR1
2. AGTR2
3. GRM2
4. GRM4
5. MTNR1A
6. OPRL1
7. Control
7/14
Redo Unsuccessful Control Digestions
SAMPLE 5' ENZYME 3' ENZYME BUFFER POSITIVE GRM4 BamH1 Xba1 2 YES HTR7A EcoR1 Xba1 2 NO GPR132 EcoR1 Xho1 2 YES EDG1 BamH1 Xho1 2 NO RO2 Xma1 none 4 NO Rs1.3 Not1 none 3 NO hM4D Nhe1 none 2 YES hM2D Xma1 none 4 NO Rs1 HindIII none 2 YES
Dump unsuccessful control samples, either we retransform or repick colonies.
7/13
Miniprep/Aar1 digestion of hM4D, Rs1.3, YFP, SSF
Digestion#2 (CONTROL DIGEST)
PLASMID 5' ENZYME 3' ENZYME INSERT SIZE BUFFER POSITIVE MNTR1A EcoR1 Xho1 1052 2 YES GRM4 BamH1 Xba1 2739 2 NO AGTR2 EcoR1 Xho1 1092 2 YES AGTR1 EcoR1 Xho1 1080 2 YES GRM2 BamH1 Xho1 2619 3 YES HTR7A EcoR1 Xba1 1338 2 NO HTR2B BamH1 Xho1 1445 3 YES V1B BamH1 Xho1 1275 3 YES CCR7 EcoR1 Xho1 1137 2 YES GPR132 EcoR1 Xho1 1143 2 NO EDG1 BamH1 Xho1 1149 3 NO EDG2 BamH1 Xho1 1095 3 YES OPRL1 EcoR1 Xho1 1113 2 YES ADRA1A EcoR1 Xho1 1401 2 YES HTR1A BamH1 Xho1 1270 3 YES Ro2 Xma1 none 1646 4 NO hM3.2 BamH1 none 944 3 YES Rs1.3 Not1 none 1448 3 NO hM2D Xma1 none 1353 4 NO hM4D Nhe1 none 1260 2 NO
7/9 Did minipreps for multiple pMAX-GFP colonies, combined all the plasmids into one microcentrifuge and the yield was 249.4ng/ul.
Made a digestion, Cathy ran a gel
Cathy, and I worked on analyzing FACS results from Tuesday and Wednesday's transwells/transfections. Analyzed data on the Flowjo program; organized the data on excel, then put the results on a bar graph.
7/8 Transwell 8 variations of the RASSL hM4. The 8 were L1, L2, L3, M1, M2, H1, H2, and H3
Chemoattractant Dilutions CNO (0nM, 10nM, 100nM, 1uM)
7/7 Transfection (Contransfection) Add two constructs/vectors into our HL-60 cell line (RASSL + pMAXGFP) Use 5 days HL-60 differentiated with DMSO
Transfected hM2, hM3, hM4, Rs1.3, all including pMAXGFP into HL-60 cells Incubate in 37C for 4 hours. Used for transwell assay.
CNO Dilution is 10nM fMLP Dilution is 10nM
7/6 We used Amaxa transfection to get pMAX GFP DNA into 4 days HL-60 cells
Protocol provided from: http://cellpropulsionlab.pbworks.com (copied over from limwiki.ucsf.edu)
100ml monocyte media for HL-60 cells
78ml Gibco IMDM
20ml 20% FBS
1ml Glutamate
1ml Antibiotic/Antimycotics
2.75ml Nucleofector solution
2.25ml Cell Line Nucleofector Solution V
.50ml Supplement
Positive transfection showed cells with green fluorescence, there was about 40% fluorescence meaning 40% success rate.
We have to note that using the right centrifuge makes a difference in transfection results. Using the incorrect centrifuge resulted in lower cell count and a 30% fluorescence while the correct centrifuge resulted in a much higher cell count as well as a fluorescence of 70%.
GPCR Miniprep alpha-pix, intersectin, P-Rex 5, DOR-ERM, DOR EZRIN, B2AR, LPD 775-1250, BPRX GFP-VASP, GFP-VASP BPRX
Transformations DOR-KIFC, DOR-ACTININ, B2AR ACTININ, LPD 775-1250, ActA 30-612, ActA 225-392, Vav, and Beta-Pix
50ul Competent cells (DH5-alpha)
50ul 1x PCM (40ul water/10ul 5x PCM)
1ul Plasmid
101ul TOTAL
Plated on CarB+Amp, incubating at 37C
7/2 Cathy, Caitlin and I learned how to analyze our cells with the FACS machine. Sometimes the FACS machine is not accurate, so we add beads to increase accuracy. The machine takes about 40 seconds per sample, we had 56 samples.
Beads stock is 1,000,000beads/ml, we used 25ul which is 25,000 beads. If we have 100 cells and 20,000 beads shown on the FACS, then we can assume that only 80% (20,000 of 25,000) of the cells and beads are shown, So 100% of the cells would be 125 cells. I learned how to analyze these numbers on a computer program Flowjo, excel, as well as on a graph.
7/1 Practice a second protocol runthrough of Transwell assay
6/29 First protocol runthrough of Transwell assay
6/25 Ran gels and checked dictyostellium confluency
6/23-6/18 Plate dicty, run gels, do minipreps, and purify plasmids
