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