User:Ryanliang

From 2009.igem.org

(Difference between revisions)
(New page: '''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-...)
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'''8/13'''
'''8/13'''
Tranfections
Tranfections
 +
1. Car1-FRB
1. Car1-FRB
 +
2. hM4
2. hM4
 +
3. SSF-YFP-hM4D-βPix
3. SSF-YFP-hM4D-βPix
 +
4. hM4D Act A Long
4. hM4D Act A Long
 +
5. hM4D Act A Short
5. hM4D Act A Short
 +
6. hM4D LPD Short
6. hM4D LPD Short
 +
Transfection Efficiency
Transfection Efficiency
 +
Car1-FRB: 42.4%
Car1-FRB: 42.4%
 +
hM4:  42.4%
hM4:  42.4%
 +
SSF-YFP-hM4D-βPix: 40.4%
SSF-YFP-hM4D-βPix: 40.4%
 +
hM4D Act A Long: 46.4%
hM4D Act A Long: 46.4%
 +
hM4D Act A Short: 36.5%
hM4D Act A Short: 36.5%
 +
hM4D LPD Short: 44.3%
hM4D LPD Short: 44.3%
 +
Chemoattractant Dilutions
Chemoattractant Dilutions
 +
CNO [0nM, 10nM, 100nM, 1uM]
CNO [0nM, 10nM, 100nM, 1uM]
 +
cAMP [0nM, 10nM, 100nM, 1uM]
cAMP [0nM, 10nM, 100nM, 1uM]
 +
fMLP [100nM]
fMLP [100nM]
 +
Result: hM4D worked only
Result: hM4D worked only
 +
'''8/12'''
'''8/12'''
 +
Transfections
Transfections
 +
1. GPR132
1. GPR132
 +
2. LPA1
2. LPA1
 +
3. OPRL1
3. OPRL1
 +
4. Control
4. Control
 +
Transfection Efficiency:
Transfection Efficiency:
 +
GPR132: 41%
GPR132: 41%
 +
LPA1: 43.2%
LPA1: 43.2%
 +
OPRL1: 50%
OPRL1: 50%
 +
Chemoattractant Dilutions
Chemoattractant Dilutions
 +
LPC: [0nM, 10nM, 100nM, 1uM]
LPC: [0nM, 10nM, 100nM, 1uM]
 +
LPA: [0nM, 100nM, 500nM, 1uM]
LPA: [0nM, 100nM, 500nM, 1uM]
 +
Orphanin FQ: [0nM, 1nM, 10nM, 100nM]
Orphanin FQ: [0nM, 1nM, 10nM, 100nM]
 +
fMLP [100nM]
fMLP [100nM]
 +
RESULTS: OPRL1 worked only
RESULTS: OPRL1 worked only
 +
'''8/10'''
'''8/10'''
 +
 +
Transfections
Transfections
 +
1. DOR
1. DOR
 +
2. DOR ACTININ
2. DOR ACTININ
 +
3. DOR ERM
3. DOR ERM
 +
4. DOR EZRIN
4. DOR EZRIN
 +
5. DOR KIFC
5. DOR KIFC
 +
6. DOR VHEAD
6. DOR VHEAD
 +
7. hM2D
7. hM2D
 +
8. hM3D
8. hM3D
 +
9. hM3.2D
9. hM3.2D
 +
10. Control
10. Control
 +
Transfection Efficiency:
Transfection Efficiency:
 +
DOR: 40.9%
DOR: 40.9%
 +
DOR ACTININ: 39.5%
DOR ACTININ: 39.5%
 +
DOR ERM: 47.5%
DOR ERM: 47.5%
 +
DOR EZRIN: 48.1%
DOR EZRIN: 48.1%
 +
DOR KIFC: 39.2%
DOR KIFC: 39.2%
 +
DOR VHEAD: 32.1%
DOR VHEAD: 32.1%
 +
hM2D: 35.2%
hM2D: 35.2%
 +
hM3D: 51%
hM3D: 51%
 +
hM3.2D: 54.7%
hM3.2D: 54.7%
 +
Chemoattractant Dilutions
Chemoattractant Dilutions
 +
DADLE [0nM, 1nM, 10nM, 100nM]
DADLE [0nM, 1nM, 10nM, 100nM]
 +
CNO [0nM, 10nM, 100nM, 1uM]
CNO [0nM, 10nM, 100nM, 1uM]
 +
fMLP [100nM]
fMLP [100nM]
 +
Results: DOR, DOR ACTININ, DOR EZRIN, DOR KIFC, DOR VHEAD, and hM3.2D worked
Results: DOR, DOR ACTININ, DOR EZRIN, DOR KIFC, DOR VHEAD, and hM3.2D worked
 +
'''8/6'''
'''8/6'''
 +
 +
Transfections
Transfections
 +
1. ADRA1A
1. ADRA1A
 +
2. EDG1
2. EDG1
 +
3. GRM2
3. GRM2
 +
4. GRM4
4. GRM4
 +
5. MTNR1A
5. MTNR1A
 +
6. OPRL1
6. OPRL1
 +
7. VIB
7. VIB
 +
8. Control
8. Control
 +
Chemoattractant Dilutions
Chemoattractant Dilutions
 +
Epinephrine: [0nM, 10nM, 100nM, 1000nM]
Epinephrine: [0nM, 10nM, 100nM, 1000nM]
 +
S1P: [0nM, 10nM, 100nM, 1uM]
S1P: [0nM, 10nM, 100nM, 1uM]
 +
Glutamate: [0nM, 10nM, 100nM, 1uM]
Glutamate: [0nM, 10nM, 100nM, 1uM]
 +
CNO [0nM, 10nM, 100nM, 1uM]
CNO [0nM, 10nM, 100nM, 1uM]
 +
Melatonin: [0nM, 1nM, 10nM, 1uM]
Melatonin: [0nM, 1nM, 10nM, 1uM]
 +
Orphanin FQ: [0nM, 1nM, 10nM, 100nM]
Orphanin FQ: [0nM, 1nM, 10nM, 100nM]
 +
Vasopressin [0nM, 10nM, 100nM, 1uM]
Vasopressin [0nM, 10nM, 100nM, 1uM]
 +
fMLP: [100nM]
fMLP: [100nM]
 +
'''8/5'''
'''8/5'''
 +
Transfections
Transfections
 +
1. CCR7
1. CCR7
 +
2. DOR
2. DOR
 +
3. DOR ERM
3. DOR ERM
 +
4. DOR EZRIN
4. DOR EZRIN
 +
5. DOR KIFC
5. DOR KIFC
 +
6. DOR VHEAD
6. DOR VHEAD
 +
7. hM3
7. hM3
 +
8. Hm3.2
8. Hm3.2
 +
9. Control
9. Control
 +
Transfection Efficiency:
Transfection Efficiency:
 +
CCR7: 45.2%
CCR7: 45.2%
 +
DOR: 62.8%
DOR: 62.8%
 +
DOR ERM: 45.7%
DOR ERM: 45.7%
 +
DOR EZRIN: 40%
DOR EZRIN: 40%
 +
DOR KIFC: 35.2%
DOR KIFC: 35.2%
 +
DOR VHEAD: 42.4%
DOR VHEAD: 42.4%
 +
hM3: 47.4%
hM3: 47.4%
 +
hM3.2: 30.9%
hM3.2: 30.9%
 +
Chemoattractant Dilutions:
Chemoattractant Dilutions:
 +
MIP-3Beta (0ug/ml, 0.01ug/ml, 0.1ug/ml, 1ug/ml)
MIP-3Beta (0ug/ml, 0.01ug/ml, 0.1ug/ml, 1ug/ml)
 +
DADLE [0nM, 10nM, 100nM, 1uM]
DADLE [0nM, 10nM, 100nM, 1uM]
 +
CNO [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.
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'''
'''8.4'''
 +
Transfections
Transfections
 +
1.ADRA1A
1.ADRA1A
 +
2.EDG1
2.EDG1
 +
3.GPR132
3.GPR132
 +
4.GRM2
4.GRM2
 +
5.GRM4
5.GRM4
 +
6.hM3
6.hM3
 +
7.LPA1
7.LPA1
 +
8.MTNR1A
8.MTNR1A
 +
9.OPRL1
9.OPRL1
 +
10.V1B
10.V1B
 +
11. Control
11. Control
 +
Chemoattractant Dilutions
Chemoattractant Dilutions
 +
Epinephrine: [0nM, 10nM, 100nM, 1000nM]
Epinephrine: [0nM, 10nM, 100nM, 1000nM]
 +
S1P: [0nM, 10nM, 100nM, 1uM]
S1P: [0nM, 10nM, 100nM, 1uM]
 +
LPC: [0nM, 10nM, 100nM, 1uM]
LPC: [0nM, 10nM, 100nM, 1uM]
 +
Glutamate: [0nM, 10nM, 100nM, 1uM]
Glutamate: [0nM, 10nM, 100nM, 1uM]
 +
CNO [0nM, 10nM, 100nM, 1uM]
CNO [0nM, 10nM, 100nM, 1uM]
 +
LPA: [0nM, 100nM, 500nM, 1uM]
LPA: [0nM, 100nM, 500nM, 1uM]
 +
Melatonin: [0nM, 1nM, 10nM, 1uM]
Melatonin: [0nM, 1nM, 10nM, 1uM]
 +
Orphanin FQ: [0nM, 1nM, 10nM, 100nM]
Orphanin FQ: [0nM, 1nM, 10nM, 100nM]
 +
Vasopressin [0nM, 10nM, 100nM, 1uM]
Vasopressin [0nM, 10nM, 100nM, 1uM]
 +
fMLP: [100nM]
fMLP: [100nM]
 +
RESULTS: None, machine broke, redo 8/6
RESULTS: None, machine broke, redo 8/6
 +
'''8/3'''
'''8/3'''
 +
Transfections
Transfections
 +
1. AGTR1
1. AGTR1
 +
2. AGTR2
2. AGTR2
 +
3. B2AR
3. B2AR
 +
4. B2AR EZRIN
4. B2AR EZRIN
 +
5. hM3.2
5. hM3.2
 +
6. hM4
6. hM4
 +
7. HTR1A
7. HTR1A
 +
8. HTR2B
8. HTR2B
 +
9. HTR7A
9. HTR7A
 +
10. Rs1
10. Rs1
 +
11. Rs1.3
11. Rs1.3
 +
12. Control
12. Control
 +
Chemoattractant Dilutions:
Chemoattractant Dilutions:
 +
Isoprenaline [0nM, 1nM, 10nM, 100nM]
Isoprenaline [0nM, 1nM, 10nM, 100nM]
 +
CNO: [0nM, 10nM, 100nM, 1uM]
CNO: [0nM, 10nM, 100nM, 1uM]
 +
Zacopride: [0nM, 10nM, 100nM, 1uM]
Zacopride: [0nM, 10nM, 100nM, 1uM]
 +
Angiontensin II (0nM, 1nM, 10nM, 100nM)
Angiontensin II (0nM, 1nM, 10nM, 100nM)
 +
Seratonin (0nM, 10nM, 100nM, 1000nM)
Seratonin (0nM, 10nM, 100nM, 1000nM)
 +
Glutamate [0nM, 10nM, 100nM, 1uM]
Glutamate [0nM, 10nM, 100nM, 1uM]
 +
fMLP: [100nM]
fMLP: [100nM]
 +
'''7/31'''
'''7/31'''
 +
Goal: Combine WT cells and GPCR cells in the same well/insert only if we can give WT RFP.
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
1. Transfection with DsRed2 and mCherry
 +
2. Dye cells with Red Vybrant @ 0,1,2,5,15,20 minutes
2. Dye cells with Red Vybrant @ 0,1,2,5,15,20 minutes
 +
DsRed2 and mCherry takes too long to show RFP (5+ hours)
DsRed2 and mCherry takes too long to show RFP (5+ hours)
 +
Cells are dyed at the optimum amount by 15minutes.
Cells are dyed at the optimum amount by 15minutes.
 +
'''7/29'''
'''7/29'''
 +
Transwell
Transwell
 +
Test new and old stocks of CNO in Millipore and BD
Test new and old stocks of CNO in Millipore and BD
 +
Chemoattractant Dilutions:
Chemoattractant Dilutions:
 +
fMLP [100nM]
fMLP [100nM]
 +
CNO [10nM, 100nM] Old & New
CNO [10nM, 100nM] Old & New
 +
'''7/28'''
'''7/28'''
 +
Transfections
Transfections
 +
hM4 Transients in Millipore, BD, and Corning
hM4 Transients in Millipore, BD, and Corning
 +
Chemoattractant Dilutions:
Chemoattractant Dilutions:
 +
CNO [0nM, 10nM, 100nM, 1uM]
CNO [0nM, 10nM, 100nM, 1uM]
 +
fMLP [10mM]
fMLP [10mM]
 +
RESULTS: BD is best
RESULTS: BD is best
 +
'''7/27'''
'''7/27'''
 +
Site DIrected Mutagenesis of DOCK insert in pTOPO
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
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'''
'''7/21'''
 +
Transfections
Transfections
 +
1. ADRA1A
1. ADRA1A
 +
2. B2AR
2. B2AR
 +
3. B2AR EZRIN
3. B2AR EZRIN
 +
4. HTR1A
4. HTR1A
 +
5. HTR2B
5. HTR2B
 +
6. GPR132
6. GPR132
 +
7. LPA1
7. LPA1
 +
8. Control
8. Control
 +
Chemoattractant Dilutions:
Chemoattractant Dilutions:
 +
Epinephrine (0nM, 10nM, 100nM, 1000nM)
Epinephrine (0nM, 10nM, 100nM, 1000nM)
 +
Isoprenaline(0nM, 100pM, 1nM, 10nM)
Isoprenaline(0nM, 100pM, 1nM, 10nM)
 +
LPA (0nM, 100nM, 500nM, 1uM)
LPA (0nM, 100nM, 500nM, 1uM)
 +
LPC (0nM, 10nM, 100nM, 1uM)
LPC (0nM, 10nM, 100nM, 1uM)
 +
Serotonin (0nM, 10nM, 100nM, 1000nM)
Serotonin (0nM, 10nM, 100nM, 1000nM)
 +
'''7/20'''
'''7/20'''
 +
Transfections
Transfections
 +
1. AGTR1
1. AGTR1
 +
2. AGTR2
2. AGTR2
 +
3. CCR7
3. CCR7
 +
4. GRM2
4. GRM2
 +
5. GRM4
5. GRM4
 +
6. MTNR1A
6. MTNR1A
 +
7. OPRL1
7. OPRL1
 +
8. VIB
8. VIB
 +
9. Control
9. Control
 +
Dilution Calculations
Dilution Calculations
 +
Angiontensin II (0nM, 1nM, 10nM, 100nM)
Angiontensin II (0nM, 1nM, 10nM, 100nM)
 +
MIP-3Beta (0ug/ml, 0.01ug/ml, 0.1ug/ml, 1ug/ml)
MIP-3Beta (0ug/ml, 0.01ug/ml, 0.1ug/ml, 1ug/ml)
 +
Vasopressin (0nM, 10nM, 100nM, 1uM)
Vasopressin (0nM, 10nM, 100nM, 1uM)
 +
Orphanin FQ [0nM, 1nM, 10nM, 100nM]
Orphanin FQ [0nM, 1nM, 10nM, 100nM]
 +
Glutamate [0nM, 10nM, 100nM, 1uM]
Glutamate [0nM, 10nM, 100nM, 1uM]
 +
Melatonin [0nM, 1nM, 10nM, 1uM]
Melatonin [0nM, 1nM, 10nM, 1uM]
 +
'''7/15'''
'''7/15'''
 +
Transfections
Transfections
 +
1. AGTR1
1. AGTR1
 +
2. AGTR2
2. AGTR2
 +
3. GRM2
3. GRM2
 +
4. GRM4
4. GRM4
 +
5. MTNR1A
5. MTNR1A
 +
6. OPRL1
6. OPRL1
 +
7. Control
7. Control
 +
'''7/14'''
'''7/14'''
 +
Redo Unsuccessful Control Digestions
Redo Unsuccessful Control Digestions
 +
  SAMPLE 5' ENZYME 3' ENZYME BUFFER POSITIVE
  SAMPLE 5' ENZYME 3' ENZYME BUFFER POSITIVE
-
  GRM4 BamH1 Xba1 2 YES
+
  GRM4 BamH1               Xba1 2 YES
-
  HTR7A EcoR1 Xba1 2 NO
+
  HTR7A EcoR1               Xba1 2 NO
-
  GPR132 EcoR1 Xho1 2 YES
+
  GPR132 EcoR1               Xho1 2 YES
-
  EDG1 BamH1 Xho1 2 NO
+
  EDG1 BamH1               Xho1 2 NO
-
  RO2 Xma1 none 4 NO
+
  RO2 Xma1               none 4 NO
-
  Rs1.3 Not1 none 3 NO
+
  Rs1.3 Not1               none 3 NO
-
  hM4D Nhe1 none 2 YES
+
  hM4D Nhe1               none 2 YES
-
  hM2D Xma1 none 4 NO
+
  hM2D Xma1               none 4 NO
-
  Rs1 HindIII none 2 YES
+
  Rs1 HindIII             none 2 YES
 +
 
Dump unsuccessful control samples, either we retransform or repick colonies.
Dump unsuccessful control samples, either we retransform or repick colonies.
 +
'''7/13'''
'''7/13'''
 +
Miniprep/Aar1 digestion of hM4D, Rs1.3, YFP, SSF
Miniprep/Aar1 digestion of hM4D, Rs1.3, YFP, SSF
 +
Digestion#2 (CONTROL DIGEST)
Digestion#2 (CONTROL DIGEST)
  PLASMID 5' ENZYME 3' ENZYME INSERT SIZE BUFFER POSITIVE
  PLASMID 5' ENZYME 3' ENZYME INSERT SIZE BUFFER POSITIVE
-
  MNTR1A EcoR1 Xho1 1052 2 YES
+
  MNTR1A             EcoR1     Xho1       1052 2 YES
-
  GRM4 BamH1 Xba1 2739 2 NO
+
  GRM4             BamH1     Xba1       2739 2 NO
-
  AGTR2 EcoR1 Xho1 1092 2 YES
+
  AGTR2             EcoR1     Xho1       1092 2 YES
-
  AGTR1 EcoR1 Xho1 1080 2 YES
+
  AGTR1             EcoR1     Xho1       1080 2 YES
-
  GRM2 BamH1 Xho1 2619 3 YES
+
  GRM2             BamH1     Xho1       2619 3 YES
-
  HTR7A EcoR1 Xba1 1338 2 NO
+
  HTR7A             EcoR1     Xba1       1338 2 NO
-
  HTR2B BamH1   Xho1 1445 3 YES
+
  HTR2B             BamH1           Xho1       1445 3 YES
-
  V1B BamH1 Xho1 1275 3 YES
+
  V1B             BamH1       Xho1       1275 3 YES
-
  CCR7 EcoR1 Xho1 1137 2 YES
+
  CCR7             EcoR1     Xho1       1137 2 YES
-
  GPR132 EcoR1 Xho1 1143 2 NO
+
  GPR132             EcoR1     Xho1       1143 2 NO
-
  EDG1 BamH1 Xho1 1149 3 NO
+
  EDG1             BamH1     Xho1       1149 3 NO
-
  EDG2 BamH1 Xho1 1095 3 YES
+
  EDG2             BamH1     Xho1       1095 3 YES
-
  OPRL1 EcoR1 Xho1 1113 2 YES
+
  OPRL1             EcoR1     Xho1       1113 2 YES
-
  ADRA1A EcoR1 Xho1 1401 2 YES
+
  ADRA1A             EcoR1     Xho1       1401 2 YES
-
  HTR1A BamH1 Xho1 1270 3 YES
+
  HTR1A             BamH1     Xho1       1270 3 YES
-
  Ro2 Xma1 none   1646 4 NO
+
  Ro2             Xma1     none             1646 4 NO
-
  hM3.2 BamH1 none 944 3 YES
+
  hM3.2             BamH1     none       944 3 YES
-
  Rs1.3 Not1 none 1448 3 NO
+
  Rs1.3             Not1     none       1448 3 NO
-
  hM2D Xma1 none 1353 4 NO
+
  hM2D             Xma1     none       1353 4 NO
-
  hM4D Nhe1 none 1260 2 NO
+
  hM4D             Nhe1     none       1260 2 NO
'''7/9'''
'''7/9'''
Line 332: Line 558:
78ml Gibco IMDM
78ml Gibco IMDM
20ml 20% FBS
20ml 20% FBS
-
1ml Glutamate
+
1ml Glutamate
-
1ml Antibiotic/Antimycotics
+
1ml Antibiotic/Antimycotics
2.75ml Nucleofector solution
2.75ml Nucleofector solution
2.25ml Cell Line Nucleofector Solution V
2.25ml Cell Line Nucleofector Solution V
-
.50ml Supplement
+
.50ml Supplement
Positive transfection showed cells with green fluorescence, there was about 40% fluorescence meaning 40% success rate.
Positive transfection showed cells with green fluorescence, there was about 40% fluorescence meaning 40% success rate.
Line 358: Line 584:
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.
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.
The machine takes about 40 seconds per sample, we had 56 samples.
-
 
-
WITHOUT BEADS
 
-
WITH BEADS
 
-
100ul cells
 
-
100ul cells
 
-
100ul fixation buffer
 
-
100ul fixation buffer
 
-
25ul medium 
 
-
25ul beads
 
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225ul total
 
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225ul total
 
Beads stock is 1,000,000beads/ml, we used 25ul which is 25,000 beads.
Beads stock is 1,000,000beads/ml, we used 25ul which is 25,000 beads.

Revision as of 00:39, 13 October 2009

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

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