User:Ryanliang

From 2009.igem.org

Revision as of 00:28, 13 October 2009 by Ryanliang (Talk | contribs)
(diff) ← Older revision | Latest revision (diff) | Newer revision → (diff)

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.

WITHOUT BEADS

WITH BEADS

100ul cells

100ul cells

100ul fixation buffer

100ul fixation buffer

25ul medium   

25ul beads

225ul total

225ul total

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